Download Oligochaeta: Glossoscolecidae

Phylogeny and Clitellar Morphology of the Giant
Amazonian Earthworm, Rhinodrilus priollii
(Oligochaeta: Glossoscolecidae)
Author(s): Shirley A. Lang, Marcos V. Garcia, Samuel W. James,
Charlene W. Sayers, and Daniel H. Shain
Source: The American Midland Naturalist, 167(2):384-395. 2012.
Published By: University of Notre Dame
DOI: http://dx.doi.org/10.1674/0003-0031-167.2.384
URL: http://www.bioone.org/doi/full/10.1674/0003-0031-167.2.384
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Am. Midl. Nat. (2012) 167:384–395
Phylogeny and Clitellar Morphology of the Giant Amazonian
Earthworm, Rhinodrilus priollii
(Oligochaeta: Glossoscolecidae)
SHIRLEY A. LANG
Biology Department, 315 Penn Street, Rutgers The State University of New Jersey, Camden 08102
MARCOS V. GARCIA
Embrapa Amazonia Ocidental, C.P. 319, 69.001-970 Manaus/AM, Brazil
SAMUEL W. JAMES
Biodiversity Institute, Kansas University, Lawrence 66045
CHARLENE W. SAYERS
Biology Department, 315 Penn Street, Rutgers The State University of New Jersey, Camden 08102
AND
DANIEL H. SHAIN1
Biology Department, 315 Penn Street, Rutgers The State University of New Jersey, Camden 08102
ABSTRACT.—The giant earthworm, Rhinodrilus priollii Righi 1967, is among the largest
terrestrial invertebrates known worldwide, reaching lengths .2 m. To investigate the
evolutionary history of the species and aspects of their reproductive biology, we collected R.
priollii specimens from several field sites in central Amazonia. Phylogenetic analyses of 16
individuals using a fragment of cytochrome c oxidase subunit 1 (CO1) identified seven haplotypes
that diverged between 2–8%. Population structures indicate episodes of gene flow between
populations and their divergence within the past 1–2 million years. Histological examination
of clitella from sexually mature specimens identified cocoon secretory cells throughout the
dorsal and dorsoventral epidermis. Unlike previously described secretory cells, those in R.
priollii contained granules with a proteinaceous core covered by external glycosylation.
Further, collagenous matrices formed the bulk of swollen clitella while albumin-secreting
cells were noticeably absent, collectively suggesting a mechanism of cocoon production
somewhat different from that described in other clitellate megadriles.
INTRODUCTION
Among ,6000 species of formally recognized terrestrial megadriles (Blakemore, 2009),
relatively few (,20) attain adult lengths exceeding 1 m. These atypically large earthworms
remain a scientific curiosity in terms of their ecology, physiology, behavior, and evolution.
Indeed, their large size poses challenges related to nutritional requirements, oxygen
transport, predation, burrowing, reproduction, and cocoon construction. Nevertheless,
giant earthworms have successfully and independently colonized a variety of habitats
worldwide. Examples include the Giant Gippsland Earthworm, Megascolides australis McCoy
1878 of southern Australia (up to ,2 m; Van Praagh, 1992), Megascolex mekongianus Cognetti
1922 from Vietnam (up to 2.9 m; Blakemore et al., 2005), Microchaetus microchaetus Rapp
1849 from South Aftrica (up to 1.8 m; Plisko, 1999), Tonoscolex birmanicus Gates 1926 from
1
Corresponding author: e-mail: [email protected]; Telephone: (856) 225-6144; FAX:
(856) 225-6312
384
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Burma (up to ,3 m in some reports; Gates, 1972), Glossoscolex giganteus Leuckart 1835/6
from South America (up to ,2.7 m; Beddard, 1895), Celeriella gigantean Benham 1906 from
New Zealand (up to 1.4 m; Lee, 1959), Driloleirus americanus Smith 1897 from Washington
State, North America (up to 1 m; in Blakemore et al., 2005), and several Rhinodrilus species
from South America, including the subject of this study, Rhinodrilus priollii Righi 1967.
More than 20 species of Rhinodrilus (family Glossoscolecidae) have been described, many
of which are native to South America (James and Brown, 2006). One of the largest species in
the genus, R. priollii, appears to be endemic to the Amazon Basin, but otherwise its
geographic range and other aspects of its biology remain mostly unexplored. In this report,
we present baseline data on the biogeography of R. priollii in central Amazonia, and
examine cellular and morphological aspects of its atypically large clitellum.
MATERIALS AND METHODS
SPECIMENS
Rhinodrilus priollii Righi 1967 specimens were collected between Sep. – Dec. 2008, from field
sites near Manaus, Brazil. Worms were maintained in 500–1000 L fiberglass storage vessels,
containing 2–3 cm of ground soil and forest litter from the collection site. Containers were
kept moist with occasional water sprinkling, covered, and placed in a shady area (e.g., under
forest canopy). Under such conditions, specimens survived up to several months at low
densities (e.g., 2–5 worms per container); higher densities significantly reduced survival time.
HISTOLOGY
Specimens were fixed in the field with 10% formalin for 1–3 d, then transferred to 70%
EtOH for long-term storage. Following an ascending EtOH series to xylene, worm fragments
were embedded in Paraplast X-tra (McCormick Scientific) and sectioned at 5–7 mm on a
Spencer ‘‘820’’ microtome. Sections were stained with Masson’s trichrome according to
Sheehan and Hrapchak (1980). Images were captured on a Canon Rebel XT coupled to a
Zeiss Universal with a PI 232 mm 28 adaptor (Perspective Image LLC, Beaverton, OR).
DNA EXTRACTION AND AMPLIFICATION
Tissue samples (,30 mg of muscle scraped from inside the cuticle) from postmortem
specimens (fixed in 70% EtOH only) were removed with a scalpel, and genomic DNA was
extracted using an E.Z.N.A. Tissue Isolation Kit (Omega Bio-Tek). Mitochondrial cytochrome
c oxidase subunit 1 (CO1) was amplified from genomic DNA using universal primers LCO and
HCO as described (Folmer et al., 1994), amplifying a ,600 bp fragment.
DNA SEQUENCING AND EDITING
PCR products were excised from 1% agarose gels and prepared for sequencing using
GeneClean (MP Biomedicals, LLC). DNA sequencing was conducted by GeneWiz Inc. (South
Plainfield, New Jersey) with forward and reverse PCR primers. Sequences were viewed and
manually adjusted in ChromasPro (Technelysium, Queensland, Australia), and aligned with
CLUSTALW (Thompson et al., 1994) to determine overlapping regions suitable for
phylogenetic analyses. GenBank accession numbers for new COI sequences are listed in
corresponding Figure legends.
PHYLOGENY
Maximum-likelihood (ML) analyses were performed for all DNA comparisons, using the
pipeline sequence MUSCLE (Edgar, 2004) to align corresponding sequences from multiple
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FIG. 1.—Live Rhinodrilus priollii specimen (,2 m) held by co-author M. Garcia
individuals or homologous DNA across species, Gblocks (Castresana, 2000) for alignment
curation, PhyML (Guindon and Gascuel, 2003) for tree building and TreeDyn (Chevenet
et al., 2006) for tree drawing, as configured in the Phylogeny.fr platform (Dereeper et al.,
2008). The aLRT statistical test (approximation of the standard Likelihood Ratio Test;
Anisimova and Grascuel, 2006) embedded in PhyML determined branch support values.
Default settings were used for all parameters.
RESULTS
Specimens of Rhinodrilus (Fig. 1) were collected from four geographic locations proximal
to Manaus, Brazil: Universidade Federal do Amazonas (UFAM) experimental farm
(02u38944.350S, 60u02944.200W), Instituto Nacional de Pesquisas Amazonia (INPA) field
station (02u37932.940S, 60u02938.720W), RPPN field site near Encontro das Aguas
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LANG ET AL.: GIANT BRAZILIAN EARTHWORM
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(03906.882S, 599W 54.2919W), and a private farm (02u549330S, 59u569340W)—all below
elevation 100 m (Fig. 2). In total, ,50 specimens were collected, most of which (,30) were
from the UFAM experimental farm. Worms were generally not observed during daylight
hours except during persistent heavy rains, upon which they surfaced at densities up to
,100 per km2. Under such conditions, they moved actively atop the forest litter for the
duration of rainfall and several hours thereafter, leaving characteristic linear trails in soft
sediment (e.g., dirt roads). Occasionally, worms were observed being carried passively in
transient runoff streams during particularly heavy rainfall. Worms also surfaced during the
evening based on remnant tracks, even in the absence of rainfall (though track numbers
were much fewer under dry conditions). Specimens ranging in length between 1–2 m in an
extended position were common in undisturbed areas (e.g., primary growth forest);
developed areas, including nature reserves within urban areas, appeared to lack any
significant numbers of R. priollii individuals. The largest specimens were collected at the
UFAM field size, reaching lengths over 2 m (up to ,220 cm when extended).
PHYLOGENY
Gene fragments from CO1 were successfully amplified from 16 Rhinodrilus priollii
individuals representing seven haplotypes (Table 1); seven specimens collected from the
PM field site identified two haplotypes that were likely Rhinodrilus contortus Cernosvitov 1938,
based on geographic and morphological criteria.
Comparisons of representative CO1 sequences indicated that haplotypes occurred within
and between populations (Fig. 3). The two major Rhinodrilus priollii haplotype lineages were
7–8% divergent in the CO1 locus, and contained representatives from multiple populations
suggesting recent episodes of gene flow. Two R. contortus haplotypes formed a third clade
,18% divergent from R. priollii CO1 sequences.
CLITELLAR MORPHOLOGY
The clitellum of Rhinodrilus priollii specimens was pronounced in mature individuals (up
to ,3 cm in diameter), forming a saddle-like structure around the dorso-lateral aspect of
the worm. Histological sections through clitella in mature individuals revealed thick,
collagenous layers (2–3 mm) separating outer, circular muscle fibers and the epidermis
(Fig. 4). Collagen appeared to be supported by a proteinaceous scaffolding (Fig. 5A), and
was absent in comparable body wall sections or clitella from immature individuals (Fig. 5B).
Thicker regions of the clitellum (i.e., more dorsal) contained two collagenous layers
demarcated by a relatively thin, proteinaceous boundary (Fig. 6).
Granulated secretory cells were observed throughout the dorsal and dorsolateral
epidermis, along the length of the clitellum. Cell bodies were typically anchored in the
outer collagenous layer, extending elaborate tubules to the epidermal surface (Fig. 7A).
Granules were packed within the cell body and tubules, often displaying an azocarminestaining core (red) surrounded by an alcian blue-staining surface (blue; Fig. 7B).
DISCUSSION
Our behavioral observations of Rhinodrilus priollii Righi 1967 are consistent with other
megadrile species, in that worms were more active during evening hours, and surface during
heavy rainfall. The cause of surfacing during rainfall has been debated, and may be related
to increased carbon dioxide levels, burrow flooding and/or mating behavior (Edwards,
2004). That no R. priollii specimens were observed at peak daylight hours suggests they are
efficient burrowers, even in the relatively hard, root-dense soil typical of central Amazonia.
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TABLE 1.—Description of field sites and number of Rhinodrilus specimens examined
Field site
Instituto Nacional de Pesquisas Amazonia (INPA)
RPPN (near Encontro das Aguas)
Universidade Federal do Amazonas (UFAM)
experimental farm
Private farm (PF)
Geographic
coordinates
Number
examined
CO1
haplotypes
02u379330S
60u029390W
03u069530S
59u549170W
02u389440S
60u029440W
02u549330S
59u569340W
5
3
2
1
9
3
7
2
Once might reasonably assume that the climatic regime and nutrient-poor soil characteristic
of equatorial Amazonia would not be suitable for a large, burrowing annelid. However, the
large size of individuals (300–400 g) likely acts as a thermal mass that prolongs survival in
exposed areas; for example, relatively small specimens of Rhinodrilus priollii and other worm
species were often found desiccated on dirt roads, while large specimens were not. More
importantly perhaps is that lack of soil nutrients may necessitate ingestion and processing of
large soil quantities to meet nutritional requirements. Indeed, dissected specimens were filled
with soil debris, though gut: body ratios appear comparable to common earthworms, (e.g.,
Lumbricus terrestris. Eisenia fetida). Gigantism in worms may, in principle, represent an
evolutionary advantage in nutrient-deficient soils by permitting enhanced nutrient absorption
due to a longer gut, but experimental support for this notion is lacking (see below).
CLITELLUM MORPHOLOGY
In addition to their unusual length, a striking morphological feature of mature
Rhinodrilus priollii specimens is their saddle-like clitellum which can measure up to ,3 cm
in diameter, almost twice the diameter of a typical midbody segment. Histological sections
revealed the underlying source of this biomass, namely collagenous layers that separated
outer muscle layers from the epidermis. These collagenous matrices are likely transient
structures associated with reproduction, based on their absence in clitella of immature
specimens or midbody segments. Note that comparable collagenous material has not been
described in other earthworm clitella; rather, elongated albumen-secreting cells appear in a
similar position and are major contributors to clitellum swelling (Grove, 1925; Lufty, 1965).
Possibly, R. priollii specimens examined in this study had recently secreted cocoons thus
explaining the absence of albumen-secreting cells, though the abundance of granulated
Type II/III-like cells throughout the clitellar epidermis does not support this hypothesis
(Grove and Cowley, 1927; Sayers et al., 2009). Alternatively, albumenotrophic glands may be
positioned elsewhere in the reproductive system (e.g., prostates; Omodeo, 2000). A detailed
examination of secreted R. priollii cocoons and reproductive structures may reveal the basis
of this apparent morphological difference.
r
FIG. 2.—Map showing field site locations near Manaus, Brazil. Major roads, BR174 and AM-010, are
indicated. INPA – Instituto Nacional de Pesquisas Amazonia; UFAM – Universidade Federal do
Amazonas experimental farm; PF – private farm; RPPN – proximal to Encontro das Aguas. North is up
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FIG. 3.—Phylogeny of cytochrome c oxidase subunit 1 (CO1) haplotypes. Two major Rhinodrilus priollii
lineages were resolved (INPA 1/ UFAM 1 and remaining haploytpes) that diverged by ,8% at the CO1
locus. Specimens collected at PF (private farm) were designated as R. contortus Cernosvitov 1938.
Numbers in parentheses indicate sample size for each haplotype. The tree was rooted with Enchytraeus
albidus CO1 (GenBank accession GU453370.1). Accession numbers for remaining sequences are:
INPA1-3 (JF501460–JF501462); UFAM1-3 (JF501463-JF501465); PF1,2 (JF501458, JF501459);
RPPN (GU014169)
FIG. 4.—Representative transverse section of the mid-clitellar region in a sexually mature Rhinodrilus
priollii specimen. Collagenous connective tissue comprised the bulk of the biomass between epidermal
and muscle layers. One half of the complete section is shown (arrow indicates the dorsal midline). Scale
bar 1 mm
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FIG. 5.—Comparison of clitellum sections between sexually mature and immature Rhinodrilus priolli
specimens. (A) Sexually mature specimens displayed thick layers of collagenous tissue (Col) supported
by proteinaceous scaffolding (arrowheads). Epidermal (Ep) and circular muscle (CM) layers were
positioned on either side. Scale bar 200 mm. (B) Sexually immature specimen lacking connective tissue.
LM – longitudinal muscle. Scale bar 50 mm
In principle, collagenous layers observed in the Rhinodrilus priollii clitellum may have dual
functions. First, the volumetric expansion of the clitellum leads to a linear increase in
epidermal surface area, thus providing additional physical space for cocoon secretory glands.
Secondly, the diameter of the secreted cocoon sheath will consequently be larger than the
body, thus facilitating cocoon deposition via its characteristic sliding over smaller, anterior
segments to remove the cocoon sheath (see Stephenson, 1930; Coleman and Shain, 2009).
Secretory glands observed in peripheral regions of the collagenous layer and epidermis
were morphologically similar to Type II/III cells reported previously in the leech,
Theromyzon tessulatum (Sayers et al., 2009). In contrast to those cells, however, Rhinodrilus
priollii glands displayed granules that stained with both alcian-blue (outer granule) and
azocarmine (inner granule) in Masson’s stain, suggesting a proteinaceous core surrounded
by a glycosylated external layer. In T. tessulatum, Type II cells (glycosylated) and Type III
cells (proteinaceous) give rise to opercula and cocoon membrane structures, respectively
(Sayers et al., 2009). Thus, the apparent fusion of Type II and Type III granules observed in
R. priollii suggests a different mechanism of cocoon construction that will require further
investigation. Note that histological staining of comparable cells in other species using
hematoxylin and eosin (H&E) and other stains did not resolve the morphologically
indistinguishable Type II and Type III cells (blue and red in Masson’s stain, respectively;
Sayers et al., 2009), and thus meaningful comparisons are difficult to make between R. priollii
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FIG. 6.—Dorsal regions of the Rhinodrilus priollii clitellum contained multiple collagenous layers.
In addition to proteinaceous scaffolding (arrowheads), a thicker proteinaceous boundary (arrow)
separated collagenous (Col) layers. CM – circular muscle, LM – longitudinal muscle. Scale bar 500 mm
and cocoon secretory cells reported in older literature (e.g., Grove and Cowley, 1927;
Defretin and Demailly, 1953; Lufty, 1965; Fleming and Baron, 1982).
EVOLUTIONARY CONSIDERATIONS
Using CO1 molecular clock variance values (e.g., Knowlton et al., 1993; Chang et al., 2008), the
relative depth of Rhinodrilus priollii populations based on CO1 haplotype divergence (,8%)
suggests they have occupied the Amazon Basin over the past 1–2 million years. Rhinodrilus priollii
individuals are capable of moving considerable distances based on deep structures detected
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FIG. 7.—Granulated secretory cells populated the dorsal and dorsolateral epidermis of the Rhinodrilus
priollii clitellum. (A) Granules were abundant in cell bodies (arrowheads) and tubules (asterisks) that
extended to the epidermal (Ep) surface. Scale bar 50 mm. (B) Granules (arrows) contained an
azocarmine-staining core (red) and alcian blue-staining exterior (blue). Scale bar 20 mm
within populations (see Fig. 3). Active dispersal by crawling is the most obvious mechanism by
which R. priollii populations have expanded, but passive dispersal by transient run-off streams
(as observed in this study), and possibly in seasonal or permanent waterways, cannot be
excluded as an important dispersal mechanism. Taken together, the current geographic range
of R. priollii is likely to extend well beyond the area surveyed here, and additional sampling may
reveal an ancestry considerably older than what has been determined here.
Giant earthworms have arisen independently and on multiple occasions, based on their
higher-ranked taxonomic positions (e.g., Moniligasteridae, Microchaetidae, Glossoscolecidae,
Megascolecidae; Blakemore et al., 2005). Further, they inhabit various environments, climatic
regimes and geographic regions (e.g., Australia; Vietnam; South Africa, North America, South
America, etc.), leaving open the question as to what underlying mechanism(s) or environmental factor(s), if any, lead to gigantism. In other animals (e.g., insects, mammals), gigantism
has been explained as a consequence of elevated growth hormone levels (Nijhout, 1998;
Maheshwari et al., 2000), a phenotype that is relatively rare within Animalia. By analogy,
stochastic variation in growth hormone levels across the Clitellata as a consequence of random
genetic variance may explain the occasional and punctuated (Eldredge and Gould, 1972)
appearance of giant earthworm species in a variety of habitats worldwide.
Acknowledgments.—Supported by a Fullbright scholarship and NSF grant IBN-0417081000 to DHS.
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SUBMITTED 9 MAY 2011
ACCEPTED 4 NOVEMBER 2011