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Reconciling genealogical and morphological species in a worldwide study of the Family Hydractiniidae (Cnidaria, Hydrozoa) Blackwell Publishing Ltd MARIA PIA MIGLIETTA,* PETER SCHUCHERT & CLIFFORD W. CUNNINGHAM Submitted: 7 July 2008 Accepted: 10 November 2008 doi:10.1111/j.1463-6409.2008.00376.x Miglietta, M. P., Schuchert, P. & Cunningham, C. W. (2009). Reconciling genealogical and morphological species in a worldwide study of the Family Hydractiniidae (Cnidaria, Hydrozoa). — Zoologica Scripta, 38, 403–430. The Hydractiniidae are a family of globally distributed marine hydrozoans (class Hydrozoa, phylum Cnidaria). Despite being one of the most well-studied families of the Hydrozoa, their genus and species-level taxonomy is unsettled and disputed. The taxonomic difficulties of the Hydractiniidae are due to many inadequate species descriptions, a paucity of available morphological characters, many cryptic species, and the often-extreme plasticity seen when colonies of the same species are found at different stages of growth or different environmental conditions. This confusion over species identity is especially important because some species of the family Hydractiniidae are well-established model organisms for a wide array of studies ranging from gene expression to developmental biology and colony growth. Here we report the species-level implications of 226 mitochondrial large ribosomal subunit (16S) rDNA sequences from around the world and 52 nuclear DNA sequences (Elongation Factor 1α) with the intent to reconcile described morphospecies with genealogical lineages. Our data show that Podocoryna carnea and P. exigua are distinct and geographically disjunct species, P. borealis is paraphyletic with respect to Podocoryna sp. from South Africa and P. bella from New Zealand. Podocoryna australis, from New Zealand form a distinct monophyletic group. Podocoryna from New England, New York and Florida all fall into a distinct monophyletic group (P. americana) and fail to support the existence of a distinct, P. selena in the Gulf of Mexico. Hydractinia pruvoti is the only species within the Podocoryna clade without fully formed medusae. We identify a Clava clade closely related to other algae dwelling Hydractiniidae. Our data do not recover Stylactaria inabai from Japan as a distinct species from S. misakiensis, and S. carcinicola as distinct from H. epiconcha. Also, 10 colonies identified as S. carcinicola fall into a distantly related clade that is close to the American S. hooperi. Finally, we identify Janaria mirabilis as the sister group to the H. echinata species complex and clarify the relationships between the H. echinata, H. symbiopollicaris, H. polyclina, H. symbiolongicarpus and H. [GM]. Corresponding author: Maria Pia Miglietta, Biology Department, Pennsylvania State University, University Park, PA 16802, USA. E-mail: [email protected] Current address: Biology Department, Pennsylvania State University, University Park, PA 16802, USA Peter Schuchert, Museum d’Histoire Naturelle, CH1211, Route malagnou 1, Geneva, Switzerland. E-mail: [email protected] Clifford Cunningham, Department of Biology, Duke University, Durham, NC 27708, USA. E-mail: [email protected] Introduction The Hydractiniidae are a family of globally distributed marine hydrozoans (class Hydrozoa, phylum Cnidaria). The basic hydrozoan life cycle includes a benthic colonial stage that reproduces asexually, and a pelagic sexual medusa (jellyfish) stage that is asexually produced by the colony (Fig. 1). While some hydractiniid species retain the complete polyp/medusa life cycle, in most hydractiniid species the swimming medusa stage is reduced to the point where it is never released from the colony. The polyps of hydrozoan colonies are connected to one another by gastrovascular canals known as stolons (Fig. 1). Hydractiniid colonies typically grow on marine surfaces as epibionts, with most living on gastropod shells occupied by living gastropods or by hermit crabs (Mills 1976; Buss & Yund 1989; Schuchert 2008). Other hydractiniid colonies grow on algae or rock substrata. The family Hydractiniidae © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 403 Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. Fig. 1 A, B. General hydrozoan life cycle with fully functional medusa (typical of the genus Podocoryna, Fam. Hydractiniidae) (A) and with medusa reduced to sporosac (B). Intermediate forms, with medusae reduced to non-feeding, swimming medusoids are also found in the Hydrozoa in general and in the Family Hydractiniidae. comprises about 106 nominal species and 10 extant genera (Bouillon et al. 2006). Despite being one of the most well-studied families of the Hydrozoa, the genus and species-level taxonomy is unsettled and disputed (Schuchert 2008; Boero et al. 1998). The taxonomic difficulties of the Hydractiniidae are due both to a paucity of morphological characters, and the often-extreme plasticity seen when colonies of the same species are found at different stages of growth or different environmental conditions. Here we report the species-level implications of 226 mitochondrial large ribosomal subunit (16S) rDNA sequences from around the world. Difficulty of diagnosing hydractiniid genera The three main and most speciose genera of the Hydractiniidae (Hydractinia van Beneden 1867; Podocoryna Sars 1846; and Stylactaria Stechow 1921a) are traditionally distinguished with a combination of two morphological and developmental characters, one involving the developmental stage of sexually mature medusae, and the other involving the developmental stage of the colony itself. The likelihood that the major genera are not monophyletic led Bouillon et al. (1997), Boero et al. (1998) and Bouillon et al. (2006) to propose merging Hydractinia, Stylactaria and Podocoryna into the oldest one — Hydractinia. These authors used the argument that neither the state of the hydrorhiza nor the presence/absence of a medusa allows an unambiguous separation of the genera due 404 to intergrading forms. The present paper does not aim to resolve the genus-level discussion; this will be done in a separate study that includes more nuclear genes. Some genus allocations used in this article are thus provisional and correspond more to common usage than strict monophyletic clades. Degree of medusa reduction in hydractiniid genera The genus Podocoryna is characterized by having fully-formed swimming medusae that are asexually produced from the benthic colony stage. These medusae are normally fully formed, that is, they have radial canals, tentacles and a manubrium with a functional mouth. In all the other hydractiniid genera — including the largest genera (Stylactaria and Hydractinia) — the medusae becomes sexually mature before they complete development (paedomorphosis, Boero & Sara 1987; Cunningham & Buss 1993). There are two levels of paedomorphosis in the Hydractiniidae, eumedusoids and sporosacs. Eumedusoids are partially formed medusae that become sexually mature without completing development to the point where they can swim normally. Depending on the species or the environmental conditions, eumedusoids are sometimes released from the colony, but cannot move far before they release their gametes. In other cases, eumedusoids are never released from the colony, effectively ending the alternation of asexual and sexual generations that characterize the Hydrozoa. Sporosacs represent the most extreme case of Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae paedomorphosis, so that gametes are produced inside sacs that are not only attached to the colony, but have lost any traces of the medusa stage, most notably the radial canals that are present in eumedusoids. Characteristics of colony formation in hydractiniid genera The genera Stylactaria and Hydractinia have been diagnosed by the structure of the hydrorhiza, the network of stolons connecting the polyps (Millard 1975; Calder 1988; Hirohito 1988; Schuchert 1996). The hydrorhiza of Stylactaria is formed by stolons — often forming a close reticulated meshwork — that are always enclosed in the non-living, chitinous periderm. The hydrorhiza of Hydractinia colonies is composed of stolons that coalesce into a mat whose top layer is composed of living tissue, formally referred to as naked coenosarc. Chitin is produced in other parts of Hydractinia colonies, but not on the top layer of the mat. In many species of the genus Podocoryna — which differs from the other genera by producing free-swimming fully-formed medusae — colonies routinely progress from periderm-covered reticulate stolons to a more fully-developed coalescence of stolons with naked coenosarc. In terms of timing of development, these can be described as events of heterochrony (Blackstone 1996). The Stylactaria condition can be described as paedomorphic, since it stops at the periderm-covered stage, and the Hydractinia condition can be described as accelerated since it reaches the naked coenosarc stage well before achieving reproductive maturity (Blackstone 1996). Rare hydractiniid genera Other genera in the Hydractiniidae are Hydrissa Stechow 1921, Clavactinia Thornely 1904, Janaria Stechow 1921a, Hydrocorella Stechow 1921b, Fiordlandia Schuchert 1996, Kinetocodium Kramp 1922, Hansiella Bouillon 1980, and Tregubovia Picard 1958 (see Bouillon et al. 2006). These genera are generally rare, and morphologically very distinct. For example, Janaria and Hydrocorella form fully calcified skeletons. Finally, the genus Clava was originally placed in the Family Clavidae, but has been recently reassigned to the Hydractiniidae (Schuchert 2001). Difficulty of diagnosing hydractiniid species Species-level identification is famously difficult not only in the Hydractiniidae but in the entire class Hydrozoa. Many of the more than 100 nominal species of Hydractiniidae are at present not recognizable due to inadequate descriptions (see also Schuchert 2008). Many species are known only from the original — and sometimes very old — descriptions, some dating back more than a century and often lacking morphological information now considered important for correct identification. Adding to the confusion, these animals show extreme morphological plasticity depending on the substratum or environmental conditions. As described below, morphotypes of the same species have been described as different species, resulting in an overestimation of species diversity. On the other hand, the presence of sibling species with little or no morphological diversification leads to an underestimation of this diversity. As an example, the most thoroughly studied species of the family has usually been referred to in the literature — regardless of where it was collected — as Hydractinia echinata (Fleming 1828). This was divided into four sibling species, with H. echinata in Europe, and three morphologically very similar species in North America (H. symbiolongicarpus, H. symbiopollicaris and H. polyclina Buss & Yund 1989). Even this did not reflect the extent of the cryptic diversity. As described below, there is a fourth, undescribed Hydractinia species in the Gulf of Mexico, and the European Hydractinia echinata is itself composed of two distinct species. This confusion over species identity is especially important because some species of the family Hydractiniidae are wellestablished model organisms for a wide array of studies ranging from gene expression to developmental biology and colony growth (Yund et al. 1987; Schierwater et al. 1991; Blackstone & Buss 1993; Blackstone 1996; Cartwright et al. 1999). Given the lack of clarity and necessary details of some species description, together with the intrinsic difficulty to tell Hydractiniidae species apart, many experimental studies might have been carried out on material of dubious identity (Boero et al. 1998). An mtDNA-based analysis of worldwide collection of hydractiniid species This article investigates mitochondrial and nuclear gene genealogies for 226 Hydractiniidae colonies or medusae collected worldwide (Table 1). We report an effort to reconcile described morphospecies with monophyletic groups reconstructed using fragments of the mitochondrial large ribosomal subunit gene (16S) and the nuclear gene Elongation Factor 1α. The 16S gene is much more easily amplified in Hydrozoa than COI. Although the 16S gene evolves about one-third as fast as COI (Govindarajan et al. 2005), comparisons of several cryptic species in the hydrozoan genus Obelia did not find any cases where a monophyletic group was identified by COI but not 16S (Govindarajan et al. 2005), and the same is true for several hydractiniid species (Cunningham, unpublished observations). Where possible, we apply a genealogical species concept to provisionally identify evolutionarily significant units (Baum & Shaw 1995). Materials and methods Collection of fresh material For a complete list of the 226 samples, localities, voucher specimens, and GenBank accession numbers see Table 1. Sequences belonging to seven species of the family Stylasteridae © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 405 Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Specimen code in trees Primary species identification Collecting sites Susbstate Voucher specimen Identified by GenBank accession number 16S/EF1α 045 Clavactina gallensis 046 H. serrata USA 047 H. rubricata NZ 48b H. serrata USA 050 H. serrata USA 052 H. serrata USA 053 H. serrata USA 012 Hydractinia spec. 072 H. polyclina USA 070 H. echinata 071 H. echinata USA 074 H. symbiolongicarpus USA 073 H. symbiolongicarpus USA 075 H. symbiolongicarpus USA 065 Hydrissa sodalis Japan 066 Hydrissa sodalis Japan 067 Hydrissa sodalis Japan 068 Hydrissa sodalis Japan 069 Janaria mirabilis 077 H. altispina S. Africa 078 Hydractinia sp. California 079 S uchidai Japan 081 H. laevispina 080 H. milleri Canada 082 H. milleri USA 083 S. reticulata Japan 084 H. epiconcha Japan 085 H. epiconcha Japan 086 S. carcinicola Japan 087 S. carcinicola Japan 088 S. carcinicola Japan 089 S. carcinicola Japan 090 H. epiconcha Japan 091 S. carcinicola Japan 092 S. carcinicola Japan 093 S. carcinicola Japan 094 H. epiconcha Japan 095 H. epiconcha Japan 096 H. epiconcha Japan 097 H. epiconcha Japan 098 S. carcinicola Japan 099 S. carcinicola Japan Clavactinia gallensis Hydractinia serrata Hydractinia rubricata Hydractinia serrata Hydractinia serrata Hydractinia serrata Hydractinia serrata Hydractinia spec. 2 Hydractinia polyclina Hydractinia echinata Hydractinia echinata Hydractinia symbiolongicarpus Hydractinia symbiolongicarpus Hydractinia symbiolongicarpus Hydrissa sodalis Hydrissa sodalis Hydrissa sodalis Hydrissa sodalis Janaria mirabilis Hydractinia altispina Hydractinia sp. Stylactaria uchidai Hydractinia laevispina Hydractinia milleri Hydractinia milleri Stylactaria reticulata Hydractinia epiconcha Hydractinia epiconcha Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Hydractinia epiconcha Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Hydractinia epiconcha Hydractinia epiconcha Hydractinia epiconcha Hydractinia epiconcha Stylactaria carcinicola Stylactaria carcinicola Hua Hin, Gulf of Siam,Thailand Friday Harbor, WA, USA New Zealand Friday Harbor, WA, USA Bering Sea, AK, USA Bering Sea, AK, USA Friday Harbor, WA, USA provenience unknown Woods Hole, MA, USA Roscoff, France New York Harbor, NY, USA Woods Hole, MA, USA New York Harbor, NY, USA Beaufort, NC, USA Okushiri Is., Hokkaido, Japan Okushiri Is., Hokkaido, Japan Okushiri Is., Hokkaido, Japan Okushiri Is., Hokkaido, Japan Gulf of Mexico False Bay, South Africa Monterey Bay, CA, USA Muroran, Pacific coast of Hokkaido, Japan Catalina Island, CA, USA Vancouver, Canada Bodega Bay, CA, USA Choshi, Boso Peninsula, Japan Kominato, Boso Peninsula, Japan Hirido beach, Nakagi, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Misaki, Sagami Bay, Miura Peninsula, Japan Misaki Fish Market, Sagami Bay, Japan Kominato, Boso Peninsula, Japan Ito, Izu Peninsula, Japan Hirido beach, Nakagi, Izu Peninsula, Japan Ito, Izu Peninsula, Japan Kominato, Boso Peninsula, Japan Kominato, Boso Peninsula, Japan Hirido beach, Nakagi, Izu Peninsula, Japan Misaki Fish Market, Sagami Bay, Japan Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Ito, Izu Peninsula, Japan Gastropod Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab Hemit crab — Pagurus acadianus Hermit crab Hermit crab Pagurus longicarpus Pagurus longicarpus Pagurus longicarpus Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab Gastropod Sea weed (Codium) Hermit crab — Dock Piles Rock — Pollia mollis Pollia mollis Gastropod Turbo sp. Turbo sp. Turbo sp. Pollia mollis Turbo sp. Gastropod Gastropod Pollia mollis Gastropod Pollia mollis Turbo sp. Gastropod Gastropod MHNG INVE33470 — paratypes — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — P. Schuchert C. Cunningham P. Schuchert C. Cunningham C. Cunningham C. Cunningham C. Cunningham A. Collins L. Buss Lab. L. Buss Lab. M.P. Miglietta M.P. Miglietta M.P. Miglietta L. Buss Lab. M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta L. Buss Lab M.P. Miglietta A. Govindarajan L. Buss Lab. L. Buss Lab. A. Brinkman-Voss L. Buss Lab. H. Namikawa M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P.Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta FJ214377 xxx–xxx FJ214378 xxx-xxx FJ214594 FJ214595 FJ214597 FJ214379 xxx–xxx/FJ372856 xxx–xxx/FJ372855 FJ214556 FJ214380 FJ214552 FJ214551/FJ372857 FJ214547 FJ214548 FJ214553 FJ214554/FJ372853 FJ214555/FJ372854 FJ214381/FJ372858 FJ214382/FJ372859 FJ214383 FJ214386 FJ214385/FJ372860 FJ214384/FJ372899 FJ214387/FJ372861 FJ214388 FJ214389 FJ214390 FJ214391 FJ214392/FJ372862 FJ214393 FJ214394 FJ214395 FJ214396 FJ214397 FJ214398 FJ214399 FJ214400 FJ214401 FJ214402 FJ214403 Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. 406 Table 1 List of the vaucher specimens, primary species identification in the filed, collecting sites, substrates, GenBank accession numbers, and identifier. 407 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 Specimen code in trees Primary species identification Collecting sites Susbstate Voucher specimen Identified by GenBank accession number 16S/EF1α 100 S. carcinicola Japan 101 H. epiconcha Japan 102 H. epiconcha Japan 051 H. epiconcha Japan 104 S. carcinicola Japan 105 S. carcinicola Japan 106 S. carcinicola Japan 107 S. carcinicola Japan 108 S. carcinicola Japan 109 H. epiconcha Japan 110 S. carcinicola Japan 111 S. carcinicola Japan 112 H. epiconcha Japan 114 H. epiconcha Japan 115 H. epiconcha Japan 116 H. epiconcha Japan 117 H. epiconcha Japan 118 H. epiconcha Japan 119 S. carcinicola Japan 120 H. epiconcha Japan 121 S. carcinicola Japan 122 S. carcinicola Japan 123 S. carcinicola Japan 124 S. carcinicola Japan 125 S. carcinicola Japan 126 S. carcinicola Japan 127 H. allmani Iceland 128 Hydractinia sp. USA 129 H. antonii Aleutians 130 H. allmanii Bering Sea 131 S. conchicola Japan 132 S. conchicola Japan 133 S. conchicola Japan 134 H. fucicola Italy 001 H. inermis Italy 002 H. inermis Italy 007 H. inermis Italy 008 H. inermis Italy 135 Clava multicornis Iceland 136 Clava multicornis Iceland 137 Clava multicornis USA 138 Clava multicornis USA 139 Clava multicornis USA Stylactaria carcinicola Hydractinia epiconcha Hydractinia epiconcha Hydractinia epiconcha Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Hydractinia epiconcha Stylactaria carcinicola Stylactaria carcinicola Hydractinia epiconcha Hydractinia epiconcha Hydractinia epiconcha Hydractinia epiconcha Hydractinia epiconcha Hydractinia epiconcha Stylactaria carcinicola Hydractinia epiconcha Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Hydractinia allmanii Hydractinia sp. unidentified Hydractinia antonii Hydractinia allmanii Stylactaria conchicola Stylactaria conchicola Stylactaria conchicola Hydractinia fucicola Hydractinia inermis Hydractinia inermis Hydractinia inermis Hydractinia inermis Clava multicornis Clava multicornis Clava multicornis Clava multicornis Clava multicornis Shimoda Bay, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Hirido beach, Nakagi, Izu Peninsula, Japan Kominato, Boso Peninsula, Japan Ito, Izu Peninsula, Japan Ito, Izu Peninsula, Japan Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Ito, Izu Peninsula, Japan Hirido beach, Nakagi, Izu Peninsula, Japan Mkimoto Pearl Island, Toba, Kii Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Ito, Izu Peninsula, Japan Hirido beach, Nakagi, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Kominato, Boso Peninsula, Japan Kominato, Boso Peninsula, Japan Ito, Izu Peninsula, Japan Japan Shimoda Bay, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Ito, Izu Peninsula, Japan Ito, Izu Peninsula, Japan Ito, Izu Peninsula, Japan Ito, Izu Peninsula, Japan Keijflavic Harbour, Iceland CA, USA Aleutian Islands, AK, USA Bering Sea, AK, USA Oshoro, Japan Sea coast of Hokkaido, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Torre del Serpe, Otranto, Apulia, Italy Otranto, Apulia, Italy Otranto, Apulia, Italy Otranto, Apulia, Italy Otranto, Apulia, Italy Sangerdi vicinity, Iceland Sangerdi vicinity, Iceland Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Gastropod Bufoniella sp. Gastropod Pollia mollis Hermit crab Gastropod Turbo sp. Turbo sp. — Pollia mollis Turbo sp. Gastropod Pollia mollis Gastropod Pollia mollis Pollia mollis Gastropod Pollia mollis Gastropod Gastropod Turbo sp. — Gastropod Gastropod Gastropod Hermit crab Hermit crab Rock Rock Hermit crab Homalopoma amussitatum Homalopoma amussitatum Homalopoma amussitatum Sea weed Sea weed Sea weed Sea weed Sea weed Sea weed Sea weed Sea weed Sea weed Sea weed – – — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta A. Lindner NOAA cruise 2000 M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta S. Piraino M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta L. Buss Lab. FJ214404 FJ214405 FJ214406 FJ214407 FJ214408 FJ214409/FJ372863 FJ214410 FJ214411 FJ214412 FJ214413 FJ214414 FJ214415 FJ214416/FJ372864 FJ214417 FJ214418 FJ214419 FJ214420 FJ214421/FJ372865 FJ214422 FJ214423 FJ214424 FJ214425 FJ214426 FJ214427 FJ214428 FJ214429 FJ214430/FJ372866 FJ214431/FJ372867 FJ214432 FJ214433/FJ372868 FJ214434 FJ214435/FJ372869 FJ214436 FJ214437/FJ372870 FJ214502 FJ214544 FJ214545 FJ214546 FJ214438 FJ21443 FJ214440 FJ214441 FJ214442 M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 Table 1 Continued. Susbstate Voucher specimen Identified by GenBank accession number 16S/EF1α Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters Specimen code in trees Primary species identification Collecting sites 86 140 Clava multicornis USA Clava multicornis NY, USA Sea weed — M.P. Miglietta FJ214443 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 141 Clava multicornis Europe 142 P. americana USA 143 P. americana USA 144 P. americana USA 145 P. americana USA 146 P. americana USA 147 P. americana USA 148 P. americana USA 149 P. borealis Iceland 150 P. borealis Iceland 151 P. borealis Iceland 152 P. borealis Iceland 153 P. borealis Iceland 154 P. borealis Iceland 155 P. borealis Iceland 156 P. borealis 157 P. borealis Iceland 158 P. borealis Scotland 159 P. bella New Zealand 160 Podocoryna spec. 161 P. borealis Iceland 162 P. borealis Iceland 163 P. australis New Zealand 164 P. australis New Zealand 165 P. australis New Zealand 166 P. carnea Denmark 167 P. exigua France 168 P. exigua France 169 P. exigua France 170 P. exigua France 171 P. exigua France 172 P. exigua France 173 P. exigua Italy 174 P. exigua France 175 P. exigua France 176 P. exigua France 177 P. exigua France 178 P. exigua France 179 P. exigua France 180 P. exigua France P. selena Clava multicornis Podocoryna americana Podocoryna americana Podocoryna americana Podocoryna americana Podocoryna americana Podocoryna americana Podocoryna americana Podocoryna borealis Podocoryna borealis Podocoryna borealis Podocoryna borealis Podocoryna borealis Podocoryna borealis Podocoryna borealis Podocoryna borealis Podocoryna borealis Podocoryna borealis Podocoryna bella Podocoryna spec. SA Podocoryna borealis Podocoryna borealis Podocoryna australis Podocoryna australis Podocoryna australis Podocoryna carnea Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua Podocoryna exigua P. selena Europe Woods Hole, MA, USA Long Island, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Keijflavic Harbour, Iceland Keijflavic Harbour, Iceland Keijflavic Harbour, Iceland Keijflavic Harbour, Iceland Keijflavic Harbour, Iceland Keijflavic Harbour, Iceland Keijflavic Harbour, Iceland Keijflavic Harbour, Iceland Aquarium, Sangerdi Marine Lab., Iceland Dunstaffnage, Scotland Otago, New Zealand Kalk Bay, South Africa Aquarium, Sangerdi Marine Lab., Iceland Keflavic Harbour, Iceland Otago, New Zealand New Zealand New Zealand Denmark Banyuls, France Banyuls, France Banyuls, France Banyuls, France Roscoff, France Mediterranean, France Otranto, Apulia, Italy Banyuls, France Banyuls, France Banyuls, France Banyuls, France Banyuls, France Banyuls, France Banyuls, France Florida Sea weed Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab Pagurus pollicaris Pagurus longicarpus Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab — Rock *Medusae Fish *Medusae Shell Gastropod — — — — Hinia incrassata Murex brandaris Hinia incrassata Hinia incrassata — — — Murex brandaris Murex brandaris Hinia incrassata Hinia incrassata Barnacles Hinia incrassata Hinia incrassata — — — — — — — — — — — — — — — — — — Identical to AY787878 — — — — — material Schuchert (1996) material Schuchert (1996) — — — — — — — — — — — — — — — — L. Buss Lab. M.P. Miglietta L. Buss Lab. M.P. Miglietta L. Buss Lab. M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta P. Schuchert M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta P. Schuchert P.Schuchert L. Buss Lab. P. Schuchert P. Schuchert P. Schuchert P. Schuchert L. Buss Lab. L. Buss Lab. F. Boero P. Schuchert P. Schuchert P. Schuchert P. Schuchert P. Schuchert P. Schuchert P. Schuchert C. Cunningham FJ214444 FJ214445 FJ214446 FJ214448 FJ214447 FJ214449 FJ214450/FJ372903 FJ214451/FJ372904 FJ214452 FJ214453 FJ214454/FJ372871 FJ214455 FJ214456 FJ214457 FJ214458 FJ214459/FJ372872 FJ214460 FJ214461 FJ214462/FJ372873 FJ214463/FJ372874 FJ214464 FJ214465 FJ214466/FJ372875 FJ214467 FJ214468/FJ372876 FJ214469/FJ372877 FJ214470 FJ214471 FJ214472 FJ214473 FJ214474 FJ214475 FJ214476/FJ372878 FJ214477 FJ214478 FJ214479 FJ214480 FJ214481 FJ214482 FJ214483 xxx—xxx Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. 408 Table 1 Continued. 409 Specimen code in trees Primary species identification Collecting sites Susbstate Voucher specimen Identified by GenBank accession number 16S/EF1α 128 129 130 181 P. exigua France 182 H. pruvoti France 183 P. hayamaensis Japan Podocoryna exigua Hydractinia pruvoti Podocoryna hayamaensis Banyuls, France Banyuls, France Ushimado, Seto Inland Sea, Japan Hermit crab Hermit crab Crab — MHNG INVE32973 — P. Schuchert P. Schuchert M.P. Miglietta FJ214484 FJ214485/FJ372879 FJ214486 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 184 P. hayamaensis Japan 185 P. hayamaensis Japan 186 P. hayamaensis Japan 187 P. hayamaensis Japan 188 P. hayamaensis Japan 189 P. hayamaensis Japan 190 P. hayamaensis Japan 005 Stylactaria sp. Japan 192 P. hayamaensis Japan 193 P. hayamaensis Japan 194 P. hayamaensis Japan 196 Hydractinia sp. Ushimado 197 S. hooperi California 198 S. carcinicola Japan 199 S. carcinicola Japan 200 S. carcinicola Japan 201 S. carcinicola Japan Ito 202 S. carcinicola Japan 203 Hydractina cf. calderi Italy 204 S. carcinicola Japan 205 Stylactaria hooperi USA 206 S. carcinicola Japan 207 Hydractina cf. calderi Italy 208 S. carcinicola Japan 209 S. carcinicola Japan 210 S. carcinicola Japan 211 S. misakiensis Ushimado 212 S. multigranosi Japan 213 S. misakiensis Japan 214 S. misakiensis Japan 215 S. misakiensis Japan 216 S. misakiensis Japan 217 S. multigranosi Japan 218 S. multigranosi Japan 219 S. multigranosi Japan 220 S. misakiensis Japan 221 S. multigranosi Japan 222 S. misakiensis Japan 223 S. misakiensis Japan 225 S. multigranosi Japan Podocoryna hayamaensis Podocoryna hayamaensis Podocoryna hayamaensis Podocoryna hayamaensis Podocoryna hayamaensis Podocoryna hayamaensis Podocoryna hayamaensis Stylactaria sp. 1 on Sargassum Podocoryna hayamaensis Podocoryna hayamaensis Podocoryna hayamaensis Hydractina sp. Stylactaria hooperi sp. 2 Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Stylactaria carcinicola Hydractina cf. calderi Stylactaria carcinicola Stylactaria hooperi sp. 1 Stylactaria carcinicola sp. 3 Hydractina cf. calderi Stylactaria carcinicola sp. 3 Stylactaria carcinicola sp. 3 Stylactaria carcinicola sp. 3 Stylactaria misakiensis Stylactaria multigranosi Stylactaria misakiensis Stylactaria misakiensis Stylactaria misakiensis Stylactaria misakiensis Stylactaria multigranosi Stylactaria multigranosi Stylactaria multigranosi Stylactaris misakiensis Stylactaria multigranosi Stylactaria misakiensis Stylactaria misakiensis Stylactaria multigranosi Ushimado, Seto Inland Sea, Japan Shimoda Bay, Izu Peninsula, Japan Ushimado, Seto Inland Sea, Japan Ushimado, Seto Inland Sea, Japan Ushimado, Seto Inland Sea, Japan Ushimado, Seto Inland Sea, Japan Ushimado, Seto Inland Sea, Japan Kominato, Boso Peninsula, Japan Ushimado, Seto Inland Sea, Japan Ushimado, Seto Inland Sea, Japan Ushimado, Seto Inland Sea, Japan Ushimado, Seto Inland Sea, Japan Monterey Bay, CA, USA Hirido beach, Nakagi, Izu Peninsula, Japan Kominato, Boso Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Ito, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Otranto, Apulia, Italy Shimoda Bay, Izu Peninsula, Japan Woods Hole, MA, USA Ito, Izu Peninsula, Japan Otranto, Apulia, Italy Ito, Izu Peninsula, Japan Loc. unknown, Japan Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Nishiwaki beach, Ushimado, Seto Inland Sea, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Kashino, Ushimado, Seto Inland Sea, Japan Shimoda Bay, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Nishiwaki beach, Ushimado, Seto Inland Sea, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Shimoda Bay, Izu Peninsula, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Shimoda Bay, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Hermit crab Crab Hermit crab Crab Crab Hermit crab Crab Sargassum sp. Hermit crab Hermit crab Hermit crab Gastropod Gastropod Gastropod Hermit crab Rope (in aquarium) Crab Crab Gastropod — Dead shell Hermit crab Gastropod — Crab Turbo sp. Gastropod Gastropod Gastropod — — Gastropod Nassarius multigranosus Nassarius multigranosus Nassarius multigranosus — Nassarius multigranosus — — Nassarius multigranosus — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta Yakko Hirano M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta L. Buss Lab. M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta FJ214487 FJ214488 FJ214489 FJ214490 FJ214491 FJ214492/FJ372880 FJ214493/FJ372902 FJ214494 FJ214495 FJ214496 FJ214497/FJ372901 FJ214498/FJ372882 FJ214499/FJ372881 FJ214500 FJ214501/FJ372883 FJ214503/FJ372884 FJ214504 FJ214505 FJ214506 FJ214507/FJ372885 FJ214508/FJ372886 FJ214509 FJ214510/FJ372887 FJ214511 FJ214512 FJ214513 FJ214514/FJ372888 FJ214515 FJ214516 FJ214517 FJ214518 FJ214519/FJ372889 FJ214520/FJ372890 FJ214521/FJ372891 FJ214522 FJ214523 FJ214524/FJ372892 FJ214525 FJ214526/FJ372893 FJ214528 M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 Table 1 Continued. Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 Specimen code in trees Primary species identification Collecting sites Susbstate Voucher specimen Identified by GenBank accession number 16S/EF1α 224 S. inabai Japan 226 S inabai Japan 227 S. inabai Japan 228 S inabai Japan 229 S. misakiensis Japan 230 S. sp. Japan 231 S. inabai Japan 232 S. misakiensis Japan 233 S. inabai Japan 234 S. misakiensis Japan 235 S. misakiensis Japan 237 S. inabai Japan 238 S. misakiensis Japan 239 S. multigranosi Japan 240 S. sp. Japan 241 S. multigranosi Japan 071 H. echinata 029 H. symbiopollicaris USA 026 H. symbiopollicaris USA 043 Hydractinia Gulf of Mexico 044 Hydractinia Gulf of Mexico 009 H. symbiopollicaris USA 030 H. symbiopollicaris USA 172 H. echinata Denmark 173 H. echinata Denmark 045 Hydractinia Gulf of Mexico 012 H. echinata Belgium 011 H. echinata Belgium 010 H. echinata Belgium 017 H. echinata Belgium 015 H. echinata Belgium 038 H. polyclina USA 038 H. polyclina USA 040 H. symbiolongicarpus USA 041 H. symbiolongicarpus USA 042 H. symbiolongicarpus USA 020 H. symbiopollicaris USA 021 H. symbiopollicaris USA 033 H. symbiopollicaris USA 013 H. symbiopollicaris USA 034 H. symbiopollicaris USA 032 H. symbiopollicaris USA Stylactaria inabai Stylactaria inabai Stylactaria inabai Stylactaria inabai Stylactaria misakiensis Stylactaria inabai Stylactaria inabai Stylactaria misakiensis Stylactaria inabai Stylactaria misakiensis Stylactaria misakiensis Stylactaria inabai Stylactaria misakiensis Stylactaria multigranosi Stylactaria multigranosi Stylactaria multigranosi Hydractinia echinata Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia new spec. Hydractinia new spec. Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia echinata Hydractinia echinata Hydractinia new spec. Hydractinia echinata Hydractinia echinata Hydractinia echinata Hydractinia echinata Hydractinia echinata Hydractinia polyclina Hydractinia polyclina Hydractinia symbiolongicarpus Hydractinia symbiolongicarpus Hydractinia symbiolongicarpus Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia symbiopollicaris Shimoda Bay, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Misaki, Sagami Bay, Miura Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Nabeta, Shimoda Bay, Japan Misaki, Sagami Bay, Miura Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Misaki, Sagami Bay, Miura Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Shimoda Bay, Izu Peninsula, Japan Kashino, Ushimado, Seto Island Sea, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Oshoro, Japan Sea coast of Hokkaido, Japan, Scotland Woods Hole, MA, USA Woods Hole, MA, USA Panacea, Gulf of Mexico, FL, USA Panacea, Gurf of Mexico, FL, USA Woods Hole, MA, USA Woods Hole, MA, USA Fredrikshavn, Denmark Fredrikshavn, Denmark Panacea, Gurf of Mexico, FL, USA Belgium Belgium Belgium Belgium Belgium Maine, USA Maine, USA Long Island, USA Long Island, USA Long Island, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA — Gastropod Gastropod Gastropod Gastropod Hermit crab Crab — Gastropod — Gastropod Hermit crab Gastropod Nassarius multigranosus Nassarius multigranosus Nassarius multigranosus Hermit crab Pagurus pollicaris Pagurus pollicaris Hermit crab Hermit crab Pagurus pollicaris Pagurus pollicaris Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab Hermit crab Pagurus acadianus Pagurus acadianus Pagurus longicarpus Pagurus longicarpus Pagurus longicarpus Pagurus pollicaris Pagurus pollicaris Pagurus pollicaris Pagurus pollicaris Pagurus pollicaris Pagurus pollicaris — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta M.P. Miglietta L.A. Henry C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham FJ214527/FJ372894 FJ214529 FJ214530 FJ214531/FJ372895 FJ214532 FJ214533 FJ214534 FJ214535 FJ214536 FJ214537/FJ372896 FJ214538 FJ214539 FJ214540 FJ214541 FJ214542/FJ372898 FJ214543 xxx—xxx FJ214557 FJ214558 FJ214559 FJ214560 FJ214561 FJ214592 FJ214562 FJ214563 FJ214564 FJ214565 FJ214566 FJ214567 FJ214568 FJ214569 FJ214570 xxx—xxx FJ214571 FJ214572 FJ214573 FJ214574 FJ214575 FJ214576 FJ214577 FJ214578 FJ214579/FJ372900 Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. 410 Table 1 Continued. 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 Specimen code in trees Primary species identification Collecting sites Susbstate Voucher specimen Identified by GenBank accession number 16S/EF1α 028 H. symbiopollicaris USA 034 H. symbiopollicaris USA 025 H. symbiopollicaris USA 039 H. symbiopollicaris USA 036 H. symbiopollicaris USA 019 H. symbiopollicaris USA 024 H. symbiopollicaris USA 027 H. polyclina USA 023 H. polyclina USA 035 H. polyclina USA 014 H. polyclina USA 018 H. polyclina USA 016 H. polyclina USA 030 H. polyclina USA 037 H. polyclina USA 053 Adelopora crassilabrum 052 Stylaster duchassaingi 060 Stylaster roseus 061 Stylasteridae spec. 059 Stylaster sanguineus 054 Errinopora nannecea 063 Stylaster sp. Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia symbiopollicaris Hydractinia polyclina Hydractinia polyclina Hydractinia polyclina Hydractinia polyclina Hydractinia polyclina Hydractinia polyclina Hydractinia polyclina Hydractinia polyclina Adelopora crassilabrum Stylaster duchassaingi Stylaster roseus Stylasteriidae unidet. Stylaster sanguineus Errinopora nanneca Stylantheca petrograpta Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Woods Hole, MA, USA Maine, USA Maine, USA Maine, USA Maine, USA Maine, USA Maine, USA Maine, USA Maine, USA Aramis Seamount, Norfolk Ridge, New Caledonia Bahamas Grenada, Spain Aleutian Islands, AK, USA Palau Aleutian Islands, AK, USA Race Rocks, British Columbia, Canada Pagurus pollicaris Pagurus pollicaris Pagurus pollicaris Pagurus pollicaris Pagurus pollicaris Pagurus pollicaris Pagurus pollicaris Pagurus acadianus Pagurus acadianus Pagurus acadianus Pagurus acadianus Pagurus acadianus Pagurus acadianus Pagurus acadianus Pagurus acadianus Rock Rock Rock Rock Rock Rock Rock — — — — — — — — — — — — — — — USNM1027760 — USNM1078387 — — USNM1027820 — C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham C. Cunningham A. Lindner A. Lindner A. Lindner A. Lindner A. Lindner A. Lindner A. Lindner FJ214580 xxx—xxx FJ214581 FJ214582 FJ214583 FJ214584 FJ214585 FJ214586 FJ214587 FJ214588 FJ214589 FJ214590 FJ214591 FJ214592 FJ214593 xxx—xxx xxx—xxx xxx—xxx xxx—xxx xxx—xxx xxx—xxx xxx—xxx 411 M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 Table 1 Continued. Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. were made available by A. Lindner. Samples will be deposited at the Smithsonian Institution National Museum of Natural History (NMNH, Washington, DC, USA). Some specimens are also described by Schuchert (2008) and have been deposited in the Natural History Museum of Geneva (MHNG), and others were previously deposited in the Peabody Museum at Yale University (see Appendix I for specimen voucher information). Colonies were collected by SCUBA diving, snorkelling, trawling and dredging on various habitats, and by filtering sand. Plankton nets were used to collect medusae from the plankton. Samples were fixed both in formalin (for morphological investigations) and in 95% ethanol (for molecular analyses). The species were identified using, among others, bibliographic references listed in Table 2. Where possible, the collected material was also compared with type material or other reference specimens obtained as loans from various museums and private collections (Appendix 1). A total of 38 nominal species and 6 unidentified species were sampled and sequenced. For the latter set, only one colony or one medusa was found. The available material has not allowed description of new species that would meet all the requirements of the Code of Zoological Nomenclature. However, they still represent the biodiversity of the family and were therefore kept in the analyses. Not included in the analysis were sequences from the species formerly known as Podocoryna minuta (Mayer 1900b; synonymous with Lizzia blondina Mayer 1900a) and Podocorynoides minima (Trinci 1903). Both species belong in the Family Rathkeidae (Schuchert 2007). DNA sequencing A 611-bp fragment of the mitochondrial 16S gene was amplified using primers SHA 5′-ACGGAATGAACTCAAATCATGT-3′ and SHB 5′-TCGACTGTTTACCAAAAACATA-3′ (Cunningham & Buss 1993). Amplification took place under the following PCR conditions: 1 min at 94 °C, then 35 cycles of 94 °C for 15 s, 50 °C for 1 : 30 min and 72 °C for 2 : 30 min with a final extension at 72 °C for 5 min. For some representatives of each of the major clades 231 bp of the nuclear marker Elongation Factor1α (EF1α) was also amplified and sequenced. Primers for the EF1α were designed by Lindner et al. (2008). PCR annealing temperature ranged from 55 to 60 °C depending on the species. Other PCR parameters were as described above for the mitochondrial gene. The PCR product was purified using a QIAquick spincolumn purification kit (Qiagen Inc., Valencia, CA). The purified PCR product was run on a 2% agarose gel stained with ethidium bromide to assay the quantity and quality (i.e. accessory bands) of the product. The purified PCR product was used as a template for double stranded sequencing. 412 Phylogenetic analysis The sequences were first assembled and edited using the software SEQUENCHER 3.0 (Gene Codes Corp., Ann Arbor, MI). They were then aligned using CLUSTALX (Thompson et al. 1997). All the alignments were then confirmed and edited by eye in MacClade 4.06 OS X (Maddison & Maddison 2000). A variable region of the mitochondrial 16S gene, comprising a total of 28 bp was difficult to align in the complete data set, and was removed for some analyses. The nuclear EF1α sequences also contained a highly variable intron that was deleted from the analysis. Phylogenetic analysis of the aligned sequences was performed using the maximum parsimony optimality criterion in PAUP* version 4.0b10 for Macintosh (Swofford 2001) and the maximum likelihood (ML) optimality criterion in GARLI v0.951.OsX-GUI (Zwickl 2006). Clade stability was assessed by ML bootstrap analyses (Felsenstein 1985) in GARLI (100 bootstrap replicates). The ML analyses in GARLI were performed using random starting trees and default termination conditions. The best-fit model of nucleotide substitution for each gene was a six-parameter model with invariant sites and gamma distribution. The parameters for this model were estimated by GARLI during each run. Results and discussion As described in the Introduction, gene genealogies using the mitochondrial 16S gene have been shown to reliably identify reciprocally monophyletic clades in the Hydrozoa (Schuchert 2005; Govindarajan et al. 2005; Miglietta et al. 2007; Miglietta & Lessios 2008; Moura et al. 2008). The 16S gene is less successful at recovering deeper nodes within the Hydractiniidae (Cunningham & Buss 1993). In this article, we will focus mostly on assigning monophyletic groups to nominal species based on the 16S gene. A complete study of species relationships and the monophyly of genera require additional data, and will be addressed in a separate paper (Miglietta et al. manuscript in preparation). We begin by analysing all of our 16S sequences together. Then, we discuss each of four major clades or grades in turn, bringing to bear nuclear EF1α sequences where available. The nuclear data helped confirm that nuclear and mitochondrial markers are identifying genealogically distinct species. Complete phylogenies of 16S and EF1α The complete 16S data set comprised 233 sequences (Table 1), including 226 members of the Family Hydractiniidae and 7 outgroup sequences belonging to the Stylasteridae, the sister group of the Hydractiniidae (Cairns 1983a; Cairns & Barnard 1984, 1987; Cartwright et al. in press). After removing the 28 bp variable region, the aligned sequences included 611 bp of the 16S mitochondrial gene. This region included 330 constant sites, 194 phylogenetically-informative sites, and 87 autapomorphies. Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters Species Distribution Clava multicornis (Forskal 1775) Mediterranean Sea, West and East Atlantic Ocean Ceylon; India; Seychelles Greenland; Arctic New Siberian Island; Norway; Iceland False Bay and Lambert’s Bay, South Africa Aleutian Islands, AK, USA Mediterranean Sea California, USA Northeastern Atlantic from the Arctic Seas south to NW Africa Kominato, Sagami Bay, Japan Mediterranean Sea to Brittany Clavactinia gallensis Thornely 1904 Hydractinia allmanii Bonnevie 1898 Hydractinia altispina? Hartlaub 1905 Hydractinia antonii Miglietta 2006 Hydractinia calderi? Bouillon, Medel & Peña-Cantero 1997 Hydractinia laevispina Fraser 1911 Hydractinia echinata (Fleming 1923) Hydractinia epiconcha Stechow 1907 Hydractinia fucicola (Sars 1857) Hydractinia G.M. Hydractinia milleri Torrey 1902 Localities of the species were collected in the present study Woods Hole, East USA; Europe Gulf of Siam, Thailand Reykjavík, Iceland; Behring Sea, Aleutian Islands, Alaska False Bay, South Africa Aleutian Islands, Alaska, USA Santa Caterina, Italy California, USA Scotland; Denmark; Belgium; Roscoff, France Kominato, Nagaki, Japan Torre del Serpe, Otranto, Italy 413 Hydractinia multigranosi (Namikawa 1991) Hydractinia n. sp. 1 Hydractinia n. sp. 2 Hydractinia n. sp. 3 Hydractinia n. sp. 4 Hydractinia polyclina Agassiz 1862 Hydractinia pruvoti Motz-Kossowska 1905 Gulf of Mexico, Florida, USA From British Columbia to Southern California, USA Hokkaido (Oshoro Bay), Misaki — Japan Monterey Bay (CA), USA California, USA Alaska, USA Ushimato, Seto Inland Sea, Japan Northwest Atlantic, USA Mediterranean Sea Gulf of Mexico, Florida, USA Vancouver, Canada; Bodega Bay, (CA), Friday Harbor (WA), USA Oshoro, Japan Sea coast of Hokkaido, Japan Monterey Bay (CA), USA California, USA Alaska, USA Ushimato, Seto Inland Sea, Japan Maine, Woods Hole (MA), USA Banyuls, France Hydractinia rubricata Schuchert 1996 Hydractinia serrata Fraser 1911 Hydractinia sodalis Stimpson 1858 Hydractinia symbiolongicarpus Buss & Yund 1989 Hydractinia symbiopollicaris Buss & Yund 1989 Hydractinia uchidai Yamada 1947 Kaikoura to Dunedin, New Zealand West coast of US; Greenland Sagami Bay, Japan North West Atlantic, USA North West Atlantic, USA Muroran, Hokkaido, Japan Portobello, Dunedin, New Zealand Bering Sea, Alaska; WA, USA Okushiri Is., Hokkaido, Japan Woods Hole (MA), Long Island Sound, USA Woods Hole (MA), USA Muroran, Pacific coast of Hokkaido, Japan Janaria mirabilis Stechow 1921a Podocoryna n. sp. Podocoryna americana Mayer 1900a Podocoryna australis Schuchert 1996 Podocoryna bella Hand 1961 Baja California to Panama and Fiji Kalk Bay, South Africa Northwest Atlantic North Island, New Zealand Portobello, Dunedin, New Zealand Podocoryna borealis Mayer 1900a Maine, USA; Iceland; British Isles from the Channel coast to Shetland; North Sea; southern and western Norway Baja California (CA), USA Kalk Bay, South Africa Florida; Woods Hole, Long Island, USA Leigh Marine Reserve, New Zealand Portobello Marine Lab. (in aquarium tank), Dunedin, New Zealand Reykjavík, Keflavic, Iceland References Forskal 1775; Broch 1916; Pena Cantero & García Carrascosa 2002; Bouillon et al. 2004 Thornely 1904; Annandale 1915; Millard & Bouillon 1973 Mayer 1900a; Rees 1956; Naumov 1969; Schuchert 2001 Millard 1955 Miglietta 2006 Bouillon, Medel & Pena Cantero 1997; Bouillon et al. 2004 Fraser 1911 Fleming 1923; Broch 1916; Naumov 1969; Vervoort 1972; Schuchert 2001, 2008 Stechow 1907; Hirohito 1988 Sars 1857; Allman 1872; Iwasa 1934; Motz-Kossowska 1905; Bouillon et al. 2004 Cunningham & Buss 1993 Torrey 1902; Fraser 1937 Namikawa 1991 perhaps a new species perhaps a new species perhaps a new species perhaps a new species Agassiz 1862; Buss & Yund 1989; Cunningham et al. 1991 Motz-Kosowska 1905; Bouillon et al. 2004; Iwasa 1934; Bavestrello et al. 2000 Schuchert 1996 Fraser 1911; Naumov 1969; Schuchert 2001 Stimpson 1858; Goto 1910; Stechow 1921a; Hirohito 1988 Buss & Yund 1989; Cunningham et al. 1991 Buss & Yund 1989; Cunningham et al. 1991 Yamada 1947; Bouillon, Medel & Pena Cantero 1997; Namikawa, 1994 Stechow 1921a, 1962; Cairns & Barnard 1984 perhaps a new species Mayer 1900a; Edwards 1972; Mills 1976 Schuchert 1996 Hand 1961; Kramp 1968; Schuchert 1996 Mayer 1900a; Edwards 1972; Schuchert 2001 M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 Table 2 Localities where the nominal species of Hydractiniidae herein analized were reported in previous works (and relative references), and localities were the species were collected during this work. Localities of the species were collected in the present study Yamada 1947; Namikawa et al. 1990; Bouillon et al. 1997 Sigerfoos 1899; Bouillon, Medel & Peña Cantero 1997; Pena Cantero & García Carrascosa 2002 perhaps a new species Hirohito 1988; Namikawa 1991 Allman 1871; Iwasa 1934; Bouillon, Medel & Pena Cantero 1997; Bouillon et al. 2004 Iwasa 1934; Hirohito 1988; Namikawa 1991 Hirohito 1988; Bouillon, Medel & Pena Cantero 1997 Shimoda Bay, Izu Peninsula; Sagami Bay, Japan Choshi, Boso Peninsula, Japan Mills 1976; Cunningham & Buss 1993 perhaps a new species Hiro 1939; Yamada 1959; Hirohito 1988; Namikawa 1997 Podocoryna exigua Haeckel 1880 Podocoryna hayamaensis Hirohito 1988 Mediterranean Sea and Britanny Sagami Bay, Japan Podocoryna selena Mills 1976 Stylactaria n. sp. (on Sargassum) Stylactaria carcinicola Hiro 1939 Florida, USA Kominato, Boso Peninsula, Japan Muroran and Sagami Bay, Japan Stylactaria conchicola Yamada 1947 Stylactaria hooperii sp. 1 Sigerfoos 1899 Stylactaria hooperii sp. 2 Stylactaria inabai Hirohito 1988 Stylactaria inermis Allman 1871 Muroran, Japan Coldspring Harbor, Long Island; Lloyd’s Harbor, Huntington Bay Monterey Bay (CA), USA Sagami Bay, Japan Mediterranean Sea Stylactaria misakiensis Iwasa 1934 Stylactaria reticulata Hirohito 1988 Misaki and Sagami Bay, Japan Sagami Bay, Japan The complete EF1α data set comprises 54 sequences including 52 members of the family Hydractiniidae and 2 outgroup sequences belonging to the Stylasteriidae. After removing an intron of variable length, the aligned sequences included 231 bp. This region included 134 constant sites, 86 phylogeneticallyinformative sites and 11 autapomorphies. Figure 2A–C present the maximum-likelihood phylogeny for all 233 sequences. Species assignments for reciprocally monophyletic groups were made through extensive consultation with the taxonomic literature. Figure 3 shows the EF1α phylogeny for a subset of samples. This nuclear gene was more difficult to amplify and sequence, but is remarkably congruent with the 16S phylogeny. Collection details are presented in Table 1. Information about each nominal species and relevant literature are presented in Table 2. In several cases, probable species were only identified by single sequences that were diverged from other monophyletic groups of taxa, were geographically disjunct, or were confirmed by EF1α (Figs 2A–4 ). The Podocoryna clade Figure 4 shows a 16S phylogeny of this species group that includes the 28 bp variable region excluded in the full phylogeny shown in Fig. 2. This phylogeny shows a strongly supported (100% bootstrap) group that includes all sequences from species known to produce fully formed medusae — the definition of the genus Podocoryna (see above). The genus Podocoryna is paraphyletic, since this strongly supported group also includes one species with a partially-formed, paedomorphic medusa (Hydractinia pruvoti). These species are discussed in the context of the phylogeny in Figs 3 and 4. Podocoryna hayamaensis/Hydractinia pruvoti. Podocoryna hayamaensis is the only Podocoryna species we found in Japan, despite the fact that three Podocoryna species were distinguished by Emperor Hirohito (1988) from the main location where we collected (Sagami Bay, near Shimoda Bay). Although Hirohito named only one of them, this is probably nevertheless a case of over-splitting, as Hirohito (1988) often separated new species on the basis of their substratum type. Our discovery of no other Japanese Podocoryna besides P. hayamaensis is consistent with the fact that P. hayamaensis is the only Japanese Podocoryna with a fully-described life cycle (Hirohito 1988), suggesting that the other two species distinguished by Hirohito may not be valid. Some of the P. hayamaensis colonies found in the Japanese Inland Sea at Ushimato deviated from Hirohito’s description of 20 tentacles, since the Inland Sea colonies had feeding polyps with only six to eight tentacles (localities in Table 1). However, no genetic differences were found within this species, which may mean these differences are environmentally induced. Hydractinia pruvoti is the only species in the Podocoryna clade without fully formed medusae. The gonophores of this species Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters Distribution Monterey Bay (CA), USA Shimoda Bay, Izu Peninsula; Misaki, Japan Torre del Serpe, Otranto, Italy Haeckel 1880; Bouillon et al. 2004 Hirohito 1988 Species Torre del Serpe, Otranto, Italy Shimoda Bay, Izu Peninsula; Ushimato, Seto Inland Sea, Japan Florida, USA Kominato, Boso Peninsula, Japan Ito and Shimoda, Izu Peninsula; Mikimoto Pearl Island, Toba, Kii Peninsula; Nakagi, Izu Peninsula; Japan Oshoro, Japan Sea coast of Hokkaido, Japan Woods Hole, East USA References Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. 414 Table 2 Continued. M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae Fig. 2 A–C. Maximum likelihood of phylogenetic hypothesis based on partial 16S mitochondrial gene (c. 600 bp) containing all the sequences used in this study, tree split into three sections A–C. The branch length indicator represents 0.1 substitutions per site. Members of the family Stylasteridae that were used as outgroup are identified in grey. The bracketed names on the right of the phylogeny reflect the taxonomic decisions made in this article whereas the individual sequences refer to tentative assignments after collection. © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 415 Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. Fig. 2 Continued. develop into medusoids that can be released or not, depending on the circumstances (Schuchert 2008). The eumedusoids have apparently no functional mouth, but may have tentacle rudiments. Otherwise they are similar to other Podocoryna medusae and most likely represent a very recent case of paedomorphosis (Boero & Sara 1987; Cunningham & 416 Buss 1993). As discussed by Schuchert (2008), H. pruvoti is very rare, endemic to the Western Mediterranean, and is known from only a few collections. Podocoryna carnea/P. exigua. The specific status of P. carnea is especially important because it is the type species of the Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae Fig. 2 Continued. genus, and tends to be the default name given to North Atlantic Podocoryna by non-taxonomists, such as ecologists and developmental biologists (e.g. Masuda-Nakagawa et al. 2000; Bumann & Buss 2008). Our results confirm Schuchert’s (2008) judgement that P. carnea and P. exigua are distinct, geographically disjunct species, with P. carnea ranging from Norway to Denmark, and P. exigua ranging from Brittany in France to the Mediterranean (Table 1). The type locality of P. carnea is Norway (Sars 1846), and our single sample collected from the North Sea coast of Denmark (Table 1) matched the carnea morphotype (see Schuchert 2008). In the 16S phylogeny (Fig. 4), the samples of P. exigua form a tight monophyletic group relative to the sample of P. carnea, which is 10 bp diverged from the P. exigua clade (1.6% p-distance). The © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 417 Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. Fig. 3 Maximum likelihood of phylogenetic hypothesis based on the nuclear gene EF1α containing representative of each major clade of Hydractiniidae. Numbers near the nodes indicate the bootstrap value (ML, 100 replicates). Nodes without number indicate that the bootstrap support was lower than 50%. 418 Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae Fig. 4 Maximum likelihood phylogenetic hypothesis based on partial 16S mitochondrial gene of the data set containing sequences of Podocoryna and Clava species, Hydractinia pruvoti, Hydractinia inermis and Hydractinia fucicola. Bootstrap notations are as in Fig. 3. © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 419 Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. specific status of P. carnea is also supported by its position as distinct from P. exigua in the nuclear EF1α phylogeny (Fig. 3). The importance of properly defining the species status of these species is underlined by the fact that two GenBank records of P. carnea — one 16S (Collins et al. 2005; GenBank AY512513) and one EF1α sequence (Groeger et al. 1999; GenBank AJ549292) — should both be renamed as P. exigua according to Figs 3 and 4. Podocoryna americana/P. selena. The 16S sequences of Podocoryna from New England, New York and Florida all fall into a distinct monophyletic group (Fig. 4), confirming the judgement of Edwards (1972) on the specific status of P. americana. Most studies on the East Coast of the United States by non-taxonomists have named their samples P. carnea (e.g. Cunningham & Buss 1993; Bridge et al. 2004; Bumann & Buss 2008), and should now refer to P. americana. In contrast, more extensive sampling of P. americana follows Cunningham & Buss (1993) in failing to support the existence of a distinct, P. selena in the Gulf of Mexico, suggesting that P. selena should be subsumed under P. americana. This is in contrast to the situation in Hydractinia, where the Gulf of Mexico samples are strongly diverged (see below). Podocoryna borealis/P. australis/P. bella/P. sp. We collected P. borealis from Iceland and Scotland. Both the 16S and EF1α phylogenies (Figs 3 and 4) confirm Edwards’s (1972) and Schuchert’s (2008) judgement that the distinct morphology of the P. borealis medusae sets it apart from the other North Atlantic Podocoryna species. The 16S phylogeny shows P. borealis to be a paraphyletic group (Fig. 4), although the EF1α phylogeny in Fig. 3 shows two P. borealis individuals descended from a single common ancestor. Although this may simply be a rooting problem caused by the long branches of H. pruvoti and P. hayamaensis, the paraphyly of P. borealis is confirmed by two samples from the Southern hemisphere that are nested within P. borealis in both the 16S and EF1α phylogenies (Figs 3 and 4). This includes one sample of P. bella from New Zealand from a fish, and one undescribed sample from South Africa (P. sp. Table 1). The remaining Podocoryna samples from New Zealand are P. australis, which form a distinct monophyletic group in both the 16S and EF1α phylogenies (Figs 3 and 4). The observation that P. bella from New Zealand is distinct from P. australis collected from Otago Bay confirms Schuchert’s (1996) opinion that P. bella, found growing on the pigfish Congiopodus leucopaecilius is distinct from the more common P. australis. The 16S sequence of the New Zealand P. bella sample differs only in 4 bp from the Northern P. borealis (0.5%, Fig. 4), thereby falling within the range of intraspecific variation in P. borealis (Fig. 4). The adult medusa of P. bella is unknown (Schuchert 1996) and the polyps and young 420 medusae of P. bella are very similar to P. borealis. The similarities at the molecular and morphological level of P. borealis and P. bella suggest that also the unknown adult medusa of P. bella will resemble P. borealis. An unidentifiable Podocoryna medusa was collected from South Africa (Podocoryna sp. South Africa: Figs 2A and 4). The latter medusa was an immature juvenile, had eight tentacles and was therefore indistinguishable from P. borealis of the same age. The 16S sequence of this sample was 9 bp diverged from H. borealis (Fig. 4 and 1.4%). Though further sampling is needed to confirm whether the southern taxa form genealogically-monophyletic groups, the P. borealis complex appears to form a closely related, anti-tropical clade in the cold waters of the Northern and Southern Hemispheres. One prominent Podocoryna species in the North Atlantic, P. areolata collected from Norway, could not be included in this study as its 16S sequence (GeneBank accession number AM939651) became available only after all the analyses were done. A preliminary analysis including P. areolata placed also this species safely within the Podocoryna clade (data not shown) (Figs 2A and 4). The Clava clade Clava multicornis. Clava multicornis colonies generally show the characteristics of Stylactaria, with periderm-covered stolons. The medusae are strongly paedomorphic, showing the sporosac condition of a complete absence of radial canals and never being released from the colony, so that gametes are released directly from sporosacs clustered below the feeding tentacles (Schuchert 2008 and references therein). Unlike most hydractiniids, including Podocoryna, Clava polyps are never polymorphic (Schuchert 2008 and references therein). Clava is a monotypic genus whose unusual arrangement of sporosacs originally placed them in the Family Clavidae (but see Schuchert 2001). Our sampling of Clava multicornis 16S confirms Schuchert’s (2008) judgement that it is found across the North Atlantic, including Iceland (Fig. 4). Stylactaria inermis/S. fucicola/S. sp. Sargassum. Unlike most members of the Hydractiniidae, Clava multicornis is usually found growing on fucoid algae. In the Mediterranean there are two Stylactaria species that are also found growing on algae, namely S. inermis, and S. fucicola. The former is the type species for the genus Stylactaria. Like Clava, these species can also be found on substrata such as rocks, but unlike most hydractiniids, they are never found on motile substrata such as gastropod shells occupied by living snails or hermit crabs. These two species do not share Clava’s derived morphology, and have some degree of polymorphism in their polyps. Stylactaria inermis and S. fucicola are very difficult to distinguish morphologically, a difficulty that is compounded by their being found on the same substrata (Schuchert 2008). Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae The 16S sequences of S. inermis and S. fucicola show very deep divergences. The four S. inermis sequences fall into two monophyletic groups nearly 6% apart from one another (Fig. 4). Each of the S. inermis clades is 5.6% diverged from both the Clava clade and our single S. fucicola sequence (Fig. 4), suggesting that they represent two cryptic species. Finally, a colony growing on the algae of the genus Sargassum in Japan (Stylactaria sp. 1 on Sargassum, Fig. 4) is 6.3% diverged from Clava, S. inermis, and S. fucicola. To summarize, all three algae dwelling Stylactaria — which include the type for the genus — plus Clava are between 5.6 – 6.3% diverged from one another. Although additional nuclear data are needed, these four algae dwelling species probably form a monophyletic group (Miglietta et al. in preparation). The deep divergences in these lineages sharply contrast with those separating the European Podocoryna species (between 1.5% and 2.4% from one another, and 3% diverged from the P. Americana) (Fig. 4). More nuclear data and greater sampling are needed for the algae dwellers. However, for comparison the Podocoryna species, with much smaller 16S divergences, are shown by the EF1α phylogeny in Fig. 3 to be true genealogical species (see discussion above). As a final point, it is intriguing that in both the Podocoryna and Clava clades there are divergent, predominantly Mediterranean lineages (see H. pruvoti above and in Figs 3 and 4). Even the morphologically homogeneous S. inermis/S. fucicola group has three divergent lineages with distances twice as great as between most Podocoryna species. The ages of these lineages almost certainly pre-date the Mediterranean, and may be Tethyan remnants (Figs 2A, 3 and 5). The Japanese misakiensis/inabai/multigranosi clade This Japanese species group is found in shallow waters with partially developed eumedusoids having few or no tentacles, and periderm-covered stolons. The stolons place them in the polyphyletic genus Stylactaria. This species group can be divided into three closely related, geographically disjunct genealogical species based on congruent 16S and EF1α phylogenies (Figs 3 and 5). These phylogenies show three closely related groups, corresponding to geographical regions and separated by fixed differences. The northern group can clearly be assigned to S. multigranosi based on its host (the gastropod Nassarius multigranosus) and its location on the northern Japanese island of Hokkaido (Namikawa 1991). The Central Japanese group is a mix of individuals identified according to Hirohito’s (1988) diagnosis depending on their host, with Stylactaria inabai being found on hermit crabs, and S. misakiensis being found on gastropods. The EF1α and 16S phylogenies (Figs 3 and 5) show that there are no consistent DNA sequence differences between individuals collected from gastropods and hermit crabs on sandy bottoms with eelgrass at three different locations (Shimoda Marine Laboratory, Misaki Marine Station, and Nabeta). This strongly suggests that this group of putative S. inabai and S. misakiensis individuals should be collapsed into the older S. misakiensis (Iwasa 1934) — shallow-water hydroids found on either gastropods or hermit crabs off the Pacific Coast of Central Japan. The genetic distance (p) between S. misakiensis and S. multigranosi in the mitochondrial gene is 0.5%. To the South, in the Seto Inland Sea of Japan, a third genealogical species is identified by the EF1α and 16S phylogenies (Figs 3 and 5; samples 211, 213, 216, 238). These were always small colonies found on shallow, mud-dwelling gastropods with no visible reproductive structures. The genetic distances (p) between this third clade and S. misakiensis and S. multigranosi, in the mitochondrial gene, are, respectively, 0.7% and 0.5% Hirohito (1988) observed consistent morphological differences between S. misakiensis depending on whether they were found on gastropod or hermit crab hosts. These differences appear to be induced by the substratum where they are found. For example, the hermit-crab dwellers have their periderm-covered stolons in parallel bundles that can grow past the margin of the shell as the hermit crab grows larger. The gastropod dwellers have reticulate stolons and never grow past the margin of the shell. Intra-specific, substratuminduced differences in morphology are common in hydrozoans (see Piraino et al. 1990 for a brief review). The EF1α phylogeny shows the Seto Inland Sea species to be a sister-group to the Central Japanese S. misakiensis. Since there are no morphological features on the Inland Sea colonies, they will be considered a sub-species of S. misakiensis (Figs 2B, 3 and 6). The Japanese carcinicola/epiconcha group A second group of Japanese hydractiniids found off the Pacific Coast of Central Japan (variously known as S. carcinicola and H. epiconcha) can be distinguished morphologically because the formerly discussed S. misakiensis has much smaller polyps (1–2 mm) than carcinicola/epiconcha (6–10 mm). This species group illustrates both of the major problems in hydrozoan taxonomy: splitting taxa that are not distinguishable by genetic data (as was the case with S. misakiensis vs. S. inabai, above); and the inability to morphologically distinguish distantly related clades, as described in the following discussion. More than any other species group, this one shows how the presence of periderm-free areas on the colony (naked coenosarc) — traditionally used to identify the genus Hydractinia — is a misleading character. Figure 6 shows a 16S phylogeny with H. epiconcha colonies sharing identical haplotypes with S. carcinicola. It should be noted, though, that Hirohito (1988) observed that the apparently naked coenosarc of H. epiconcha had a very thin covering of periderm, consistent with being placed in Stylactaria. Although there are two divergent mitochondrial subclades in Fig. 6, a limited number of EF1α © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 421 Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. Fig. 5 Maximum likelihood phylogenetic hypotheses based on partial 16S mitochondrial gene for the Japanese S. misakiensis and S. multigranosi. Bootstrap notations are as in Fig. 3. sequences do not support distinct genealogical species (Fig. 3). Individuals from each of the subclades had identical EF1α sequences (112 H. epiconcha Japan, 089 S carcinicola Japan) that were 3 bp diverged from a colony bearing the basal mitochondrial haplotype (105 S carcinicola Japan). For this reason all these species will be collapsed under the older name of H. epiconcha (Stechow 1907), although the generic designation will need to be revisited. More importantly, of a total of 52 colonies morphologically identified as H. epiconcha or S. carcinicola, only 42 are shown in Fig. 6 as H. epiconcha. This is because the remaining 10 colonies identified as S. carcinicola (Fig. 2B, samples 189–202, 204, 206, 208–210) fall into a distantly related clade that is 422 close to the American S. hooperi (Sigerfoos 1899; discussed in following section). These 10 colonies were morphologically indistinguishable from the 22 in Fig. 6 originally identified as S. carcinicola (Fig. 6). This is a very deep divergence, on the order of millions of years, between colonies that cannot thus far be distinguished morphologically from one another. Miscellaneous clades of Hydractinia and Stylactaria Carcinicola/hooperi/calderi group (Figs 2B and 3). A morphological resemblance between S. carcinicola and the Atlantic S. hooperi (Sigerfoos 1899) has been noted in the literature (Hirohito 1988). Although most S. carcinicola colonies (22 of 32) are now collapsed into H. epiconcha (see discussion above), Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae Fig. 6 Maximum likelihood phylogenetic hypotheses based on partial 16S mitochondrial gene for the Japanese H. carcinicola. Bootstrap notations are as in Fig. 3. 10 S carcinicola colonies fall into a clade that includes a Japanese unidentified species (Hydractinia sp. 4 from the Seto Inland Sea at Ushimado), a Mediterranean species (H. calderi), and species on the Western and Eastern Coasts of North America. Although Cunningham & Buss (1993) called the species found on gastropods in the Monterey Bay California, S. hooperi, this name must be reserved for the population represented by the individual taken from a bivalve in Woods Hole, Massachusetts (205 Stylactaria hooperi USA). In the 16S tree (Fig. 2B), the Woods Hole S. hooperi falls in a clade with the 10 S carcinicola colonies mentioned above. Although this might suggest that the Japanese colonies should be named S. hooperi, limited sampling of EF1α sequences indicates that the Japanese S. carcinicola fall outside of a group that includes the Californian Monterey Bay species, the Massachusetts S. hooperi, and the Italian H. calderi (Fig. 3). Until the New England population can be sampled in greater detail, we will retain the name S. carcinicola for the 10 Japanese colonies. Hydractinia serrata (Fig. 2B). This species is very common in the Bering Sea living on gastropods, hermit crabs, and crab carapaces. One sample has been found as far south as Friday Harbor, Washington, on a hermit crab. There is a second, diverged mitochondrial lineage in the Bering Sea (46 H. serrata © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 423 Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. USA), that probably represents a cryptic species, but we were unable to amplify EF1α from H. serrata so confirmation of a second genealogical species in the Bering Sea will have to wait. Like many other hydractiniids that live on hermit crabs, H. serrata grows beyond the lip of the gastropod shell on which it is found (See discussion of S. misakiensis/inabai above, and H. polyclina below). In H. serrata, though, this growth is taken to an extreme on the hermit crab Labidochirus splendescens. This hermit crab is always found in association with H. serrata, and inspection of the shell reveals that almost all of the ‘shell’ is formed from the H. serrata colony, with only a tiny gastropod shell at its centre (Abrams et al. 1986). Colonies of H. serrata have been collected from Norway (Cunningham, personal observation), but DNA sequences are not available to confirm the species identity. Allmanii/antonii/conchicola group (Figs 2B and 3). Also found in the Bering Sea is Hydractinia allmanii (see Naumov 1969), which is only found on hermit crabs (never on gastropods or true crabs). This species does not extend the shell of its hermit crab hosts, and is also found in Iceland (Figs 2B and 3). This species has eumedusoids (Rees 1956; Schuchert 2001) with four radial canals, as does the unusual H. antonii (Miglietta 2006), which is also found in the Bering Sea. H. antonii has a calcified skeleton that is very different from the calcified skeleton in the distantly related hydractiniid Janaria (see discussion below). In the same clade, though, is the Japanese S. conchicola, which has entirely reduced sporosac reproductive structures, and has opposite host-specificity to H. allmanii, being only found on the living gastropod Homalopoma sagarensis in Hokkaido, Japan (Yamada 1947; Namikawa et al. 1992; Hirohito 1988). To summarize, this species group is found in the coldtemperate region of the northern hemisphere, including yet another likely species from the Bering Sea, 128 H. sp. USA in Fig. 2B (H. sp. 1 in Table 2). Milleri/laevispina/uchidai/altispina group (Figs 2B and 3). Like the allmanii/conchicola group, this clade also has a mix of taxa with eumedusoids retaining radial canals, and reduced sporosacs with no trace of canals or other medusoid features. This group is found in the northern and southern hemisphere, including two species with eumedusoids (Figs 2B and 3): the algae-dwelling Japanese S. reticulata and the eumedusoidbearing South African H. altispina, collected from a living gastropod. These species are basal to a monophyletic clade with sporosacs, including the Japanese H. uchidai from consolidated sediment in Muroran, Hokkaido, the algaedwelling individual collected on algae in the Monterey Bay of California (078 Hydractinia sp. in Figs 2B and 3). The sporosac-bearing group includes H. milleri, which is common on rocks in the intertidal zone of the Central Californian 424 Pacific Coast (Bodega Bay). Comparison of 16S and EF1α indicates a second genealogical species collected on a rock in deeper waters off Catalina, California, and off of a floating dock in Vancouver Canada (Figs 2B and 3). This second milleri-like species is most likely H. laevispina (though called H. californica in Cunningham & Buss 1993). H. laevispina is known to live on non-motile substrates like rocks and barnacle shells (Fraser 1922). Further sampling of both 16S and EF1α will be required to establish the apparently overlapping ranges of H. milleri and H. laevispina (Figs 2C, 3 and 7). The echinata species group The species group that has received the most attention as a model system for laboratory studies of histo-recognition (Lange et al. 1988; Buss & Grosberg 1990; Grosberg et al. 1996; Mokady & Buss 1997; Hart & Grosberg 1999; Grosberg 2000; Cadavid et al. 2004, 1997; Müller et al. 2004; Wilson & Grosberg 2004; Nicotra & Buss 2005; Powell et al. 2007) and laboratory studies is the complex of species in the North Atlantic related to Hydractinia echinata (Fleming 1828). The most important study species have been H. symbiolongicarpus in North America (e.g. Levitan & Grosberg 1993; Grosberg et al. 1997; Hart 1997; Buss & Yund 1988), and H. echinata (e.g. Lange et al. 1989; Müller et al. 2004) in Europe. The 16S and EF1α phylogenies agree that the sister group to the H. echinata clade is Janaria mirabilis from the Gulf of California. This species has a highly calcified skeleton, but shares with the echinata group the reliance on hermit crabs as hosts (Buss & Yund 1989). The H. echinata species complex is almost entirely found living on gastropod shells occupied by hermit crabs (Buss & Yund 1989). Past efforts at distinguishing this species group include tests of reproductive isolation, morphology and allozymes (Buss & Yund 1989), DNA–DNA hybridization (Cunningham et al. 1991), and 16S DNA (Cunningham & Buss 1993). These studies found significant divergence between American and European species, and deep divergence between Hydractinia in the Gulf of Mexico and the Atlantic Coast. The current study confirms the divergence of the Gulf of Mexico species, but sampling in Belgium and Denmark has revealed a second European lineage that cannot be distinguished from two American species. Hydractinia symbiolongicarpus group. Buss & Yund (1989) recognized that the Hydractinia found on the hermit crab P. longicarpus in the Long Island Sound was distinct from the Hydractinia found on the hermit crab P. pollicaris. It was thus named H. symbiolongicarpus. Subsequent DNA–DNA hybridization and 16S phylogenies found that the sister species to H. symbiolongicarpus is an undescribed species found in the Gulf of Mexico, referred to as H. [GM] in Cunningham et al. (1991) and Cunningham & Buss (1993) and Ferrell (2004). More extensive sampling of 16S confirms that H. symbiolongicarpus Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae Fig. 7 Maximum likelihood phylogenetic hypothesis based on partial 16S mitochondrial gene of the data set containing sequences of Hydractinia echinata-like species from the North Atlantic. Bootstrap notations are as in Fig. 3. is found from Maine on down to North Carolina (Fig. 7, Table 1, samples 41– 42, 73–75), and is distinct from several samples of H. [GM] from the Gulf of Mexico (43– 45). Echinata/symbiopollicaris/polyclina group. Buss & Yund (1989) named the second species in the Long Island Sound H. symbiopollicaris after its predominant host P. pollicaris. They considered H. symbiopollicaris distinct from H. polyclina found in Maine living on a third hermit crab species — P. acadianus (Agassiz 1862) — based on the reduced mating success of these two forms. Hydractinia symbiopollicaris and H. polyclina were found by DNA–DNA hybridization and 16S to be significantly © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 425 Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al. diverged from H. echinata collected in England and France (Buss & Yund 1989). The current study has found no genetic differences between H. polyclina and H. symbiopollicaris in either the EF1α or 16S (Figs 3 and 7). Preliminary studies of COI also found no consistent differences, suggesting that the specific status of H. symbiopollicaris is in doubt, and may perhaps be subsumed under the senior name of H. polyclina L. Agassiz 1862. While the more extensive sampling in this study confirms that H. echinata in France and Scotland are distinct from all American Hydractinia species in both EF1α and 16S (Figs 3 and 7), further sampling in Belgium and Denmark (samples 010–012, 015, 017, 172, 173) has revealed populations that are genetically indistinguishable from either H. symbiopollicaris or H. polyclina (Figs 3 and 7). Until further evidence is found, we conclude that the name H. polyclina applies to a trans-Atlantic species. This trans-Atlantic H. polyclina has been previously referred to as H. symbiopollicaris when found on P. pollicaris shells in North America (Cunningham et al. 1991; Cunningham & Buss 1993) and as H. echinata along the North Sea Coast of Belgium and Denmark. Geographical distribution Of the major clades of Hydractiniidae analysed in this study, 11 are found in the Northwest Pacific (Japan), 8 in the Northeast Pacific (Canada, California, Alaska, Aleutian Islands), 6 in the Mediterranean Sea, 5 in the Northwest Atlantic, 3 in the South Pacific (New Zealand), 2 in the South Atlantic (South Africa), one in the Indian Ocean (Thailand). The North Pacific and especially Japan is the region with the highest diversity of Hydractiniidae. The global distribution of clades appears to be affected to some extent by the ability to disperse through swimming medusae. The clade composed of species with swimming medusae (genus Podocoryna) has representatives around the world, from Japan to the Antipodes, Arctic and Atlantic. It appears to have achieved this broad distribution relatively rapidly, with a fairly recent ancestor compared to most other major clades in Fig. 2. For example, the most recent ancestor of its sister clade is twice as old (the Clava/S. fucicola/S. inermis group). Even further evidence of the dispersal ability of free swimming medusae is a very recent Podocoryna species group is found in Iceland, New Zealand, and South Africa (P. borealis/ P. bella/P. sp. 1 SA). On the other hand, H. polyclina is found on both sides of the North Atlantic despite reproducing with non-swimming sporosacs and being found living on hermit crabs. This is most likely a case of rafting by the host hermit crabs on algal rafts (Wares & Cunningham 2001). Algal rafting also explains the trans-Atlantic range of Clava multicornis, which also has non-swimming sporosacs, but is usually found living directly on seaweed. Other species groups lacking swimming larvae 426 are broadly distributed geographically even in the absence of swimming medusae (e.g. the H. allmanii/S. conchicola group, the S. hooperi (S. carcinicola group)). In these cases, the main explanation for their broad range appears to be their relatively old age. Concluding remarks This study shows the efficacy of using a combination of mitochondrial and nuclear markers to begin to untangle species complexes in the morphologically plastic Hydrozoa. One immediate contribution is to identify the correct names to be used for the species most often used as model organisms for ecological, physiological and developmental studies. Most studies of Atlantic Podocoryna have referred to P. carnea. In America, most of these studies have been carried out on material that should be referred to as P. americana (e.g. Cunningham & Buss 1993; Bridge et al. 2004; Baumann & Buss 2008; Masuda-Nakagawa et al. 2000), and in Europe, the studies by Schmid’s group have been carried out on P. exigua. Because of the work of Buss & Yund (1989), material in American studies have been correctly identified as H. symbiolongicarpus or H. polyclina. In Europe, where Hydractinia is most widely used as a model organism, the situation is very complicated. An essay on H. echinata as a model organism (Frank et al. 2001) mentions that H. echinata can be obtained from laboratories in Plymouth, England, Roscoff France, and Helgoland Germany. Our study shows that these represent two different species: H. echinata (Plymouth and Roscoff) and H. polyclina (Helgoland, Germany). Few of the taxa we defined were not monophyletic, but had fixed differences between them (e.g. Stylactaria multigranosi and S. misakiensis). In another case the mitochondrial and nuclear genes did not agree in the identifications of taxa (i.e. S. hooperi from East and West California). Besides these few exceptions, the criterion of monophyly we adopted worked well in defining taxa. Finally, this work sets the stage for a generic revision of the Hydractiniidae. The type for the Family is Hydractinia echinata (Fleming 1828). While much of the phylogeny is unresolved, it appears that the closest relatives to the H. echinata clade are two morphologically very distinctive genera: Hydrissa and Janaria (the latter having a fully-calcified skeleton. This means that the proposal by Bouillon et al. (1997), Boero et al. (1998) and Bouillon et al. (2006) to collapse Hydractinia, Stylactaria and Podocoryna into the single genus of ‘Hydractinia’ is not practical without eliminating clearly distinct genera such as Janaria. This means that several new monophyletic genera will have to be named on the basis of more intensive phylogenetic analysis and more sequence data. Acknowledgements This work would have not been possible without the tremendous help of many people who assisted in the field and/or sent specimens. We are especially grateful to, H. Namikawa, Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae S. Piraino, F. Boero, A. Govindarajan, A. Lindner, D. Calder, A. Brinkmann-Voss, L.A. Henry and S. Cairns for providing samples and/or helping in the field. C. Gravili helped with bibliographic aspects of the article. 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Appendix 1 Besides the newly collected specimens (see Table 1), the following specimens from museum or private collections were examined and used to compare and identify our material. Species specimen accession code Museum Hydrissa sodalis Hydractinia cryptogonia Hydractinia granulata Hydractinia granulata Podocorella minoi Podocoryna hayamamensis colony male, 29 May 1952 prep. 60 70 prep. 7496. Sp # 1682 — Paratype sp # 1685 — Holotype Hydr. 2569. Prep 4877-4880. Hyrd. # 3795 sp# 2573. Prep. 1345-13456. Hydr. # 2620. Prep # 8094-8110 — Paratype. Hydr. # 2600. Various slides Holotype and Paratype Holotype and Paratype Hydr. 3757. Various prepared slides Hydr. # 3787 Holotyype. Prep. 88.99. Hydr. 3022# 8687 — Paratype. Various prepared slides Hydr. 3888 — Paratype, Hydr 3888 (eumeduoids), Hydr. 3888 prep 10499-10495. Hydr. 1748. Paratype. Hydr 1759 Hydr. 1723, Paratype. Hydr 1722, Paratype Prep. No 21211, 21 22. Hydr. 30017, Paratype Hydr. 1725, 1730, 1724 H 408, H 481, H 2416, H 2751, 2433, 2442 H 407, H 1823, H 1853, H 2651, H 2553, H 3503, H 87, H 88, H 97, H 123, 776R, CP 258, SAM 114B H. 3504, H 2928 H 2695, H 389, TMI 61A, TME 61 H 3503, H 2416, FAL209D H 1995 H 2356, H 2358, H 2883, H 2538, H 2357 H 503 H 1867, H 389 H 2654, H 92 H 1739, H 122 H97 CP332 Si15 USNM 25503 USNM 43525 USNM 71134 USNM 76888 USNM 70980 USNM 100198 USNM 42534 USNM 43897 USNM 89155 USNM 68775 USNM 42673 USNM 16535 C-8 C-8 L-28 L-28 L-29 Q-31 Q-31 Q-31 S-32 S-32 U-30 Santa Catalina, California, USA Bodega Bay, California, USA Coll: J. Marks Bering sea Hokkaido. Coll Shin Kubota Various Speciemens and Localities Various Speciemens and Localities Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Stylactaria halecii Stylactaria misakiensis Stylactaria spiralis Stylactaria yerii Stylactari brachiurae Stylactaria inabai Stylactaria monoon Stylactaria reticulata Stylactaria spinipapillaris Hydrocorella africana Hydractinia altispina Hydractinia caffaria Clavactinia multitentaculata Hydractinia marsupalia Hydractinia sp. Hydractinia sp. Clavactinia gallsensis Clavactinia multitentaculata Hydractinia kaffaria Cyteis nassa Hydractinia canalifera Podocoryna carnea Hydractinia polyclina Hydractinia milleri Hydractinia aggragata Hydractinia sodalis Hydractinia allmani Hydractinia bayeri Hydractinia echinata Hydractinia echinata Hydractinia sp. Hydractinia sp. Hydractinia bedofii ? Hydractinia monocarpa Hydractinia serrata Hydractinia allmani Hydractinia allmani/serrata Hydractinia allmani Hydractinia serrata Hydractinia serrata Hydractiniidae Hydractinia serrata male/female Hydractinia serrata female Hydractinia serrata Hydractinia allmani Hydractinia californica Hydractinia milleri Hydractinia serrata Hydractinia serrata Hydractinia uchidai Complete Hydractiniidae collection Complete private collection 430 Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Tschuba Museum — Japan Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Cape Town Natural History Museum — SA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Smithsonian Institute — DC — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Yale Peabody Museum — CT — USA Royal Ontario Museum — Canada Boero Laboratory — Italy Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
