Download Morphometric analyses of mixed Dactylorhiza colonies (Orchidaceae)

BotaizicalJournuloJfthe Linneun So&@ (1998), 128: 385 401. With 5 figures
Artick
ID:bt980203
Morphometric analyses of mixed Dactylorhiza
colonies (Orchidaceae) on industrial Gaste sites
in England
PETER J. A. SHAW
School of Lzji Sciences, Roehumpton Institute, Whitelands College, West Hill,
London S W 1 5 3 S N
Received October 1997; acceptedf w publication3;& 1998
Morphometric data were collected from Dac&lorhiza growing on two types of industrial waste
(pulverized fuel ash or PFA, and Leblanc process waste) during the summer of 1997. Three
species grew an PFA (0.
fuchsii, D . incamata, D. praetemksa). The same species plus D. pu7purella
grew on the Leblanc site, although on both substrates the majority of plants failed to
correspond precisely with published descriptions, introducing an element of subjectivity into
the field identifications. Principal Components Analysis and Detrended Correspondence
Analysis ordinations confirmed that textbook species descriptions corresponded to extremes
of multivariate space. Cluster Analysis failed to produce a useful resolution of the data.
Discriminant Functions Analysis initially gave the misleading result that any plant with
spotted leaves was D.fuchsii, but produced useful results after leaf spotting was removed from
the analysis. O n PFA sites hybrids appeared to be mainly D. praetemzissa x D. j%chsii (= D.
grandis) or D. praetermissa x D. incamata (= D. wintoni). The identity of hybrids on the Leblanc
site was unclear, perhaps reflecting the greater age of this site which may have allowed
extensive introgression.
0 1998 The Idnnean Society of London
ADDITIONAL KEY WORDS:-colonization
waste.
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conservation
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industrial
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. . . . . . . . . .
386
388
388
388
389
390
390
392
398
400
400
~
hybridization
CONTENTS
Introduction . . . . . . . . . . . . . .
Methods . . . . . . . . . . . . . . .
Site descriptions and plant selection . . .
Morphometric measurements
. . . . .
Characters measured . . . . . . . .
Results . . . . . . . . . . . . . . .
Preliminary data description . . . . . .
Multivariate analyses . . . . . . . .
Discussion . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . .
References . . . . . . . . . . . . . .
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0 1998 The Linnean Society of London
Sel era1 hea\s) industrial processes generate such large volumes of solid wastes
that their disposal sites become locally important landscape features (Gemmell,
1977). Such derelict land has a poor public image, but many recolonized industrial
site5 contain locally important populations of eutrophication-intolerant plants
'Box, 1993; Sham, 1994). It is well known that industrial waste sites frequently
support largc populations of orchids, especially where the waste is calcareous
(Lee 8r Grcenuood. 1976; Greenkvood & Gcmmell, 1978). At most such sites
Daciylorliizn Necker ex Nevski species are the dominant orchids, though Gjmnadenia
ionottwa (L.) R.Br. subsp. conop\ea. .hacamptr~ ~ ~ ~ r a m i d a(L.)
l i s L.C.M.Rich. and
Epzpactic spp. also occur. Xomenclature follows Stace (1 997). From the virwpoint
of conservation, the presence of orchids on industrial sites is useful since these
plants hake a high public profile and hence have been uscd as a justification for
prcxenting site de\ elopment or t o force a rescue-relocation scheme (Shaw, 1994,
1998). However, problems arise lvhen trying to name the species, especially of
Dacplorhz,-n, due to the M ell documented case of hybridization between taxa in
this genus (Bateman 8r Denholm. 1983, 1985, 1989a). Stace (1997: 974) suggests
that a representatixe sample of 5- 10 non-extreme plants should be examined
I d o r e allocating a name to n Dac{)*lor/izzapopulation, and that some individuals
ma) defy satisfactor) identification. O n e extrcme rcsponse to this variability is
shoivn b) the ivarden of Nob End Leblanc waste site, who now refuses to attempt
to identif) the thousands of Dacplorhzza on his reserve below gencric lcvel (W.
Halton, perc. comm., June 1997).
'Ydcock, Gorton 8r hlorries (1983) attempted to classify the Dactylorhzza growing
on industrial sites in hlanchester (including one re-examined in this study). They
recorded DacQlothiza incarnata (L.) Soo, D. praeteimtssa (Druce) Soo, D.purpurella
(T. & T.-L Stevenson) Sob and D.fuchsii (Druce) Sob. They explicitly avoided
measuring the many plants suspected of being hybrids, and restricted their
analyses to pol) graphs and bar charts. These limitations are unfortunate, since
an adequate description of morphological \ ariation within such populations
requires multik ariate anal! ses encompassing putative hybrids as well as standard
plants (Bateman & Denholm, 1989a,b; lVilson, 1992). An example ofthis approach
i % a 5 provided by Xndersson (1994), who used multivariate analyses to examine
the 5tructure of Dnctylorhiza traunsteinui (Saut. ex Rchb.) Sob populations in
SItcden. She concludcd that plants lvhich some orchidologists would allocate to
four different species in fact represent \.ariation M ithin one heterogenous species.
Hedrt.n i 1996a.b) used alloLc).memarkers to explore lineages within DacQlorhira,
reporting that I). zncainata and D. fuchsii showed long evolutionary separation,
but that D.p m e t c i m i m and D. purpurella appeared to be allotetraploids arising
more reccntl) from hybridization between D.zncarnata and D.fuchcii.
The aim of the work described here was to transcend the labelling of individual
I)nc@lorhzza plants, and to defcribe the fbll extent of morphometric variability within
geographicall) separated populations on different industrial wastes. Both univariate
and multikariate analyses are used to examine whether any sharp boundaries or
clear clusters can be found that could correspond to recognized species distinctions.
.Uthough there have been many multi\,ariate analyses of the morphometrics of the
genus Dactdorhzza (e.g. Bateman & Denholm, 1983, 1985, 1989a; Rndersson, 1994,
MORPH ()METRICS OF DA CTYLORHf<4
387
1995)) this is the first published work using such techniques to explore variation
within hybrid swarms specifically on industrial waste sites.
Two different industrial wastes were examined, PFA and Leblanc process waste.
PFA is the fine ash resulting from burning coal in modern power stations. It is a
well-studied material that supports clearly defined vegetational changes during its
colonization (Shaw, 1992).After the soluble salts have weathered out (typically 5- 10
years), a legume-calcicole sward develops that often supports a few orchids. Birch/
willow scrub develops and closes canopy after 20-40 years, but the remaining open
glades often contain dense hybrid swarms of Dac@ylorhiza (Shaw, 1994). Curiously,
other orchid genera tend to be absent. The technology for burning pulverized coal
dates from the 1950s, so no PFA sites are older than c. 1955.
Leblanc waste is a relic of the Leblanc chemical procedure that was widely used
to manufacture bleach, sodium carbonate (washing soda), hydrochloric acid and
other chemicals (Mathews, 1978a,b). The production of one ton of sodium carbonate
generated approximately two tons of waste, consisting of calcium sulphide mixed
with lime, carbon and unburned sulphur. Virtually no Leblanc waste has been
produced since 1900 so very few Leblanc sites now survive, all in the industrial
north-west. At the Leblanc site studied it is believed that the surface of the waste
was subsequently removed for extraction of unburned sulphur by the Mond process,
before being redumped around 1890. Over the intervening 100 years the waste
weathered down to an infertile gypsum/lime mixture with extremely low levels of
nitrogen and phosphorus (Burrows, 1995).
It was decided to apply several complementary lines of analysis to the data, to
explore the stability of analytical conclusions. In addition to standard univariate
analyses, four different multivariate techniques were applied. Two ordination
algorithms were used: Principal Components Analysis (PCA) and Detrended
Correspondence Analysis (DCA, often erroneously called DECORANA after the
computer program which performs the analysis). PCA is the most mathematically
natural ordination technique (Gauch 1982; Digby & Kempton 1987) and is
closely related to Principal Coordinates Analysis, used by Bateman & Denholm
(1983, 1985, 1989a). DCA was originally devised to simplify the interpretation
of community data (Hill & Gauch, 1980) and is probably the ordination technique
most widely used by ecologists, but has also been recommended as a taxonomic
tool (e.g. Parnell & Waldren, 1996). Cluster Analysis is routinely used both in
taxonomy and community ecology to classify individuals on the basis of multivariate attributes, although the wide choice of clustering algorithms (Kent &
Coker, 1992) and the huge number of ways that any given dendrogram can be
portrayed combine to make interpretation problematic. Discriminant Function
Analysis, otherwise known as Canonical Variates Analysis (CVA: Gittins, 1985))
requires that at least some members of a sampled population can be assigned to
pre-defined categories, and uses the multivariate attributes of these individuals
to produce linear functions by which the group membership of additional
individuals can be estimated. As such it is a logical choice when attempting to
classify problematic individuals. Jeffers (1996) applied these four multivariate
analyses to a dataset describing variation in European elms (along with logistic
regression and a genetic algorithm), and found the diversity of analyses to be
helpful in giving complementary insights into the data structure.
TABLE
1. Site dcscriptions
.Sik 1. Chrshunt ,gravel pit. [1'L3i lOY2). vc18. PFA froin Britnsdown power station was deposited into a gravcl pit
11) Iiargc in c. 1956. 11 notv dcnse l)irch/\villo\v woods, with orchids in open glades along with a diverse flora
including rls/fl- ~ n / j p i i sIfilld., &a.cininc./iin nut~/miilarinI.. and grasses. A moist site, soil pH 7.4.
,Sik 2. Rnrking ash lagoon. "&658'25), vr18. A PF.1 lagoon surrounded by 3 r n high Ilunds adjacrnt t o the
'~Ihamrs.c~~ntainiiig
I'FA dumped from Barking power station in c. 1965. Plants were moved from here to
Nottingharn xs a rescue relocation schemc In E'ehruar). 1997, and biometric data were collected in thrir new site
( i n a new PFA dump at SK456:31Y as plants were establishing. Donor site was a wet lagoon of pH 7.2, whereas
the rrcipient sitv \\.as drier and morc alkalinc [pH 8.0).
S i k 3. I h a x power station. (SE(i55280). vc6 I . A srries of six PFNLgypsum mounds (each 5 ni x 5 m x 3 m high)
wcrc waI&shcd in 1987. I n 1991 soit
rraped from C:lirsliuiit arid other orchid-bearing PFA sites, and a thin
Ia)cr .c. 0.1 mm') introduced a s an inoculum sourcc. The first three years saw establishment of halophytes and
Ityimics (Sliaw. 1996).and orchids have h v e r r d in damp areas at mound haws ever) year since 1995. Substrate
pH rnngc.; 8.3 ;piire PFA) to 7.7 !~]isi~ni-coiitaiiiin~
mounds).
4.Carrington po\z-rI wtion,
793 I), vc59. PFA dumped in c. 1975 has formed a marshy commun
?ha lat@/in I,.. Phrapiik ciro/ra/
'.) Trin. cx Strud., and S d t - scrub. Opcn areas near by support dens
o C Nlorkr/oninpt~~o-folio~~
[I..) Huds. a n d other annuals. Soil pH 7.5.
.Qt, 5. Xrih End. K o l t o n . !S1)737074). vc.59. j\n
I comprising >5 ha of Ixblanc waste dating from c. 1900,
c.ontaining Sn/i.r scrub. thousands of flor!vforh& nnd f~rrn?indpnioronopscn along and other calricoles. A dry sitc,
c l r \ a t d 'LO m ak)ove the riwrs Croal and Invell. Soil p H 7.6.
AIKI'HODS
Site descriptions and plant selection
All sites visited consisted of sizeable areas (>1 ha) of alkaline industrial wastes
(Table 1). Data were collected from four PFA dumps, of which the most intensively
studied was Cheshunt sgravel pit in the Lee Valley of Hertfordshire, where a
boardwalk has been erected to allow public access to a dense stand of Dacglorhzra.
One PFA site contained plants which had recently been translocated as part of a
rescue-relocation scheme, which could have affected their development. Lehlanc
data were collected from the largest and best known surviving Leblanc site, 'Nob
End' in Bolton, a Site of Special Scientific Interest where a 10 m thickness of Leblanc
waste has been left largely unmanaged since Victorian times.
At each site, orchids were systematically sampled from within randomly selected
3 m x 5 m areas during June 1997. A provisional identification was attempted for
each plant, based on criteria given in Rose (1 98 l), Clapham, Tutin & Moore (1 989)
and Stace (1 997). Remarkably few plants matched precisely all the key characteristics
for any given species, and the identification process involved a subjective judgement
about the de<greeof allowable variation. Plants which could not satisfactorily be
assigned to a species in the field were recorded as putative hybrids. It is important
to note that, in the context of this study, the label 'hybrid' denotes only that a
decision was made in the field that a plant could not be identified with confidence,
not that its features were intermediate between two standard species.
.\ forphometric measurements
There is a balance in any morphometric study between the number of characters
measured, the number of individuals measured in any population and number of
MORPHOMETRICS OF DACTYLORHIZA
389
populations studied (Bateman & Denholm, 1989b). In this study the number of
characters measured was kept low (23, compared with 52 used by Bateman &
Denholm, 1983, 1985, 1989a) in order to allow sampling of a greater range of sites
within one season. All characters were measured in situ without damaging the plants.
Consequently, floral dimensions are not comparable with those of Bateman & Denholm
(1983, 1985, 1989a), who worked on preserved flowers. Floral measurements were
recorded to 0.1 mm using a Sylvac digital micrometer, and were replicated on two
lowermost flowers because between-flower variation was evident in many plants. Other
measurements were recorded to an accuracy of 1 mm using a ruler. Stem solidity was
tested by non-destructive fingertip squeezing of the stem base.
Characters measured
Non$oral measurements
overall height, mm
number of flowers
stem solid/hollow
length of first leaf, mm
length to widest point of the same leaf
presence/absence of leaf spots
presence/absence of a hooded leaf tip
Floral measurements
distance from stem to tip of bract, mm
distance from stem to tip of the corresponding flower, mm
distance from neck of spur to tip of labellum, mm
depth of labellar sinus, mm
labellar width (frontal view), mm
labellar depth (lateral view), mm
spur length, mm
Ground colour (red, purple or white; and whether dark or light)
Lip pattern (spots only, spots +lines, or a solid central mass of colour).
A lipfold angle was calculated as arctanpip depth/(lip width/2)]. Prior to multivariate analysis all floral data were converted to one mean value per plant, to
remove within-plant variation. Ground colour was recoded as two variables: PURPLE
(0 if red or white, 1 if pale purple, 2 if deep purple), and RED (0 if purple or white,
1 if pale red, 2 if deep red). Two lip pattern variables were included in multivariate
analyses: lines @resent/absent) and hyperchromic @resence/absence of a large solid
central blotch).
Within-plant variation between floral characters (which were measured on two
replicate flowers per plant) was examined by a paired samples t-test. Significance of
differences between substrata and between species were tested by one-way ANOVA
where the data approximated to normality, otherwise the Kruskal-Wallis test was
used (Steel & Torrie, 1982: 544). Data were ordinated by PCA (using the Pearson
correlation matrix) and Detrended Correspondence Analysis (Hill & Gauch, 1980).
Several different Cluster Analysis algorithms were applied, and Discriminant Function
Analysis (otherwise known as Canonical Variates Analysis) was applied to all plants
P J. .I SH.\\\
1” I
’I:.u~LF.2. Formal allocation of plants
t o species hased 011 the kcys provided 1)- Rose (3981),Clapham,
‘I’utin & lloore 1989:. and Stace (19‘37). The total numl~crof plants allocated to each specics by each
kcy is gi\.c.ii, along \\-itti the plants \clrich had one or morr characteristics that mis-matched any part
~
key
143
I43
bawd on the identities allocated in the field to named (i.e. apparently non-hybrid)
plants. hlost calculations \$ereperformed by ‘SPSS for Windows’, though the cluster
aiial)ses were run on LlVSP 2.0 (Ko\.ach, 1990) and DECORANA ordination on
P(:-ORD 2.0 (hijA1 Software Design. 1995).
Preliininav data deso-ipiion
F-ield ohsen ations suggested that plants represent four species D.j~clzszz, D.
prnetemica, I). znmnakl and D.purpurella. An attempt was made to classify each plmt
pa,/
hot front it\ recorded measurements, by converting each step in published
taYonomic kc) \ into a formal logical algorithm which was applied to computerised
data. For thk purpose. the labelluni Lvas defined as reflexed if the angle of fold
e~ccedcd43 deLgrees.Three ke\s Mere used: Rose (1981), Clapham, ‘lutin & Moore
/ lW9) nnd Stace (1 997). Curiousl), feucr than half the sampled non-hybrid plants
fittrd n whole sequence of key steps perfectly (Table 2), a proportion that would
diiiost ccxrtainl) have been reduced further if additional key characteristics had been
A\ ailable for analysis. A reasonable number of plants keyed down accurately to D.
jurhtu or U . incarnnta. but ver) fekv matched D.praetermirra or D.purpurella. The
commonest problem Mas for the stem to be recorded as solid for plants which would
otherwise fit accuratcl) to the (supposedly hollow stemmed) spccies, reflecting thc
dificult\ of assessing stem hollom ness non-destructively. Consequently, for the
nna1)ses below plants \\..ill be identified as the species recorded in the field, even
thouqh this introducrs an element of subjectivity.
The distribution of putative qxcics between sites is given in Table 3. (The low
numbers recorded from site 2 reflect poor establishment after translocation, due to
drought and rabbit damage.) 4 summary of the univariate data for each named
taxon is +en in Table 4. Clear differences between species are to be expected,
4ncc plants were allocated 10 species on the basis of predefined characteristics.
(.llthough not shown, F values were calculated to test between-species differencrs
for the data in Table 1 and were significant in all cases; these should be regarded
‘11 ‘I p o d hoc description rather than a true significance tcst.)
MORPHOhIEI'RICS OF DACTYLORHI44
39 I
TABLE
3. Brcakdown of species numbers by sites
1
2
3
4
5
z
Uncertain
D. fucluii
D.praeteimicra
1). incamata
D.puipurella
30
0
7
3
0
12
11
19
4
10
46
37
23
22
15
15
c
7
60
143
Site
11
1
5
0
0
0
1
0
3
7
0
57
8
11
4
0
0
TABLE 4. Summary results for univariate descriptive statistics, given as number of plants or as
meanf SE. All lengths are in mm
Species
Total examined
Spotted leaves
Hollow stem
Lip red
Lip purple
Lip white
Lip dots only
Height
Number of flowers
1,eaf Icngth
Lip length
Folding angle
Spur length
Notch depth
Uract/flower ratio
Widest point ratio
D. incarnata
D. praetennissu
22
0
15
17
2
3
0
23
0
3
0
23
0
14
D. f u c h z
37
35
1
0
22
1.i
0
D. purpurella
Hybrids
15
46
3
16
0
0
0
15
0
0
2
43
3
10
192.00k 18.00 400.001 18.00 339.00 f25.00 166.001 12.00 312.00 k 20.00
19.30 k 2.60 36.00k 3.40
36.705 3.10 16.30.t 2.10 29.005 2.60
81.00f 6.00 134.001 5.00 98.00f 4.80 64.50f 4.80 100.20f 3.90
7.10k 0.20
6.60.t 0.20
6.00k 0.20
6.20f 0.10
5.30f 0.20
64.00 f 2.00 41.00k 3.00 29.00k 3.00 35.00f 3.00 57.00.t 3.00
5.70.t 0.20
7.20.t 0.20
6.60.t 0.10
5.2Of 0.20
6.80k 0.30
0.70f 0.10
1.90.t 0.10
0.30k 0.10
0.801 0.10
0.50+ 0.04
0.73f 0.03
0.88.t 0.03
0.85f 0.03
1.03.t 0.05
0.975 0.04
0.50 0.01
0.51 .t 0.02
0.40k 0.02
0.41 k 0.01
0.30f 0.02
+
The most distinctive taxon was D. incarnata; plants were almost invariably diminutive plants with a hollow stem, hooded, unspotted leaves widest near to the
stem, and reflexed lips (most were D. incarnata subsp. coccinea (Pugsley) Sob, the
subspecies with brick-red flowers (Bateman & Denholm, 1985; Stace, 1997)).The
remaining species were more variable. Dacplorhiza purpurella was recorded only on
the Leblanc waste site, consisting of relatively small plants with deep purple flowers
and a barely notched or entire lip. None of these plants appeared to have a hollow
stem, despite this characteristic being used in all the keys previously cited. Dacplorhiza
pruetermissa was more variable; plants had unspotted leaves, a shallow notch and a
purple lip that was often decorated with dots only (no lines or dashes). Again, many
plants that otherwise matched this species accurately were recorded as apparently
possessing a solid stem, despite a hollow stem being expected. Dacplorhiza fuchsii
exhibited the greatest range of height, and was characterized by spotted leaves and
relatively flat, deeply divided lips (often with a white ground colour).
The uncertaidhybrid plants included almost the entire spectrum of variation
recorded in the putative parents, except that none had the pure red flowers that
characterize D. incarnata subsp. coccinea. Most had purple flowers whose lip was
embellished with lines and dots, though two individuals on the Leblanc site were
r -
1 WI.E 3. S u m m a n results of chi-square contingency table analyses of morphological characteristics
of hyhrid orchids
1h
1-1
Sr
*
-
Sr
*
RK
I .K
st
St
*
-
HI
Hd
RK
I .R
*
+
Ht
*
+-
+
Nt
Hd
*
*
+
St
*
+
.\ld,rt.viations: ' - '. significant iP<O.Oj; negative association betwrrn characteristics: ' ', significant (P<O.O5)
positilc association between chara<-trristirs;* indic-ates cells o n the leading diagonal of the matrix. BK. bract ratio;
Hd. hooded lcaf tip; Ht. height; I-.\, lip angle: LR. leaf ratio: Nt. Notch (sinus) depth; Sr, spur length, St, spottcd
IC'l\.C~\.
hyperchromic (Ettlinger, 1991) with a solid central mass of colour on the lip (one
with an orange centre on a ground colour of purple, the other a purple centre on
white).
IVithin-plant variation in floral dimensions is not mentioned in standard works
on this genus, but was evident in the populations studied. Of the seven continuous
floral variables, two (labellum length and bract length) differed significantly (lY0.05)
between flowers. The tendency was for the lowermost flower to have a significantly
longer labellum and bract than the next flower up.
Univariate evidence of clustering within the hybrid plants was sought by applying
the Corner test of association to each independent pair of variables in turn (this
in\.olved converting each variable into a new bistate variable, with 1 =above median
and O=below median, entering these figures into contingency tables, and then
applying the chi-squared test). The results are summarized in Table 5, showing
seven si,;nificant results out of 36 tests (itself R 0 . 0 1 from a Poisson distribution
\kith E= 36/20 = 1.8). The associations which were found to be significant are
generally consistent with the morphometrics of the parent species. The observations
that taller plants tended to have a deep labellar sinus and poorly hooded leaves,
while spotted plants had a deeper sinus, suggest influences of D.fuchsii, whereas the
tendency for shorter plants to have leaves widest closer to the stem suggests the
parentage of D. incarnata. However, these patterns are weak, and it would be easy
to dismiss the biometric properties within the hybrid swarms as wholly random.
The Kruskal-Wallis test was applied to examine whether morphometric differences
existed between plants on the Leblanc site and PFA sites. These analyses excluded
hybrid plants (which must be assumed to be a heterogeneous grouping). The results
are summarized in Table 6, showing a trend for plants from the Leblanc site to be
$mailer than those growing in PFA. Interestingly, D.jkhsii and D.praetemisra both
tended to have less reflexed lips at the Leblanc site.
Alultivariate anabses
The dataset was ordinated by Principal Components Analysis (PCA), showing
that the first two axes (accounting for 28.7% and 19.8% of the total variance
MORPHOhIETRICS OF DACTYI.ORHI<A
393
Significant differences between plants from PFA and Lcblanc sites, assessed by the
Kruskal-Wallis test. Hybrids were excluded because they were assumed a priori to be a heterogeneous
group, and Dacplorhiza purpurella was excluded as it did not occur on PFA
TABLE
6.
~
D. incamata
D. jkchaz
Height
Number of flowers
Leaf length
Lip folding angle
Bract/flower ratio
D. praetenzrsa
*-
** *** -
*-
*-
*-
*+
Abbreviations: * K0.05; ** R0.01; '-', Lehlanc value i P F A value;
significant results are left blank.
'+',
Leblanc value >PFA value. Non-
TABLE
7 . Factor loadings from multivariate analyses. Abbreviations used: % V eigenvalues, expressed as
variance except for DCA where the raw eigenvalues are given; BR: length of bract; F L distance
from stem to tip of flower; HD: presence of a hooded leaf tip; HT: height; HY: lip hyperchromic; L1:
leaf length; L2: length to widest point of leaf; LA: lipfold angle; LD: labellar depth; LN: lines on lip;
L P vertical length of labellum; L W labellar width; N F number of flowers; PP: purpleness of ground
colour; RD: redness of ground colour; SI: depth of labellar sinus; S L spur length; SM:stem solid; S P
presence/absence of leaf spot
PCA
%V:
BR:
FL
HD:
HT:
HY:
LI:
L2:
LA:
LD:
IN:
LP
LW:
NF:
RD:
PP:
SI:
SL
SM:
SP:
DCA
CVA (PFA 1)
Axis 1
28.7
Axis 2
19.8
Axis 1
0.04
Axis 2
0.01
Axis 1
69.0
0.66
0.50
-0.46
0.77
0.03
0.79
0.71
-0.56
-0.14
-0.09
0.63
0.74
0.73
-0.59
0.33
0.45
0.35
-0.32
0.36
0.55
0.62
0.69
0.25
-0.13
0.23
0.42
0.59
0.54
-0.39
-0.03
-0.44
0.03
-0.02
0.46
-0.68
0.31
0.13
-0.70
101
124
223
- 23
142
7
86
212
171
109
117
- 13
-83
585
98
- 93
127
31 1
- 298
- 38
0.47
0.04
0.08
0.13
- 37
51
94
- 135
- 47
- 29
134
80
365
- 67
- 198
- 142
32
-61
- 129
-73
- 158
- 192
Axis 2
31.0
-0.32
-0.25
-0.15
-0.02
~~
- I .OO
-0.81
0.57
0.40
1.78
1.44
-0.97
- 1.01
0.10
0.70
0.12
0.03
0.53
0.63
-0.90
-0.06
0.61
0.43
0.49 -0.31
0.53
0.02
0.05 -0.10
0.87
0.49
-0.92
0.32
CVA (PFA 2)
CVA (Leblanc)
Axis1
62.8
Axis 2
37.2
Axis 1
58.5
Axis 2
25.4
0.45
0.82
0.27
0.67
-0.12
0.63
-0.01
0.34
-0.17
0.36
-0.44
0.80
0.26
- 0.07
0.37
-0.06
~
~
0.28
-1.16
0.78 - 0.09
2.40
0.03
0.18
- 1.47
0.41 - 0.60
0.19
-0.10
0.98
0.00
-0.89
-0.74
0.77
0.0 1
-0.14
0.33
-0.24
- 0.32
-0.05
0.06
0.16
0.00
._
~
~~
-0.87
0.78
4.07
-3.20
0.24
0.08
I .60
-0.57
1.05
-0.37
-0.25
-0.36
-0.17
~
- 1.07
0.34
- 1.99
1.41
1.31
0.18
-0.80
-0.17
-0.13
0.44
0.87
-0.56
-0.02
~
respectively) describe a roughly triangular layout of points. The factor loadings are
given in Table 7. The first axis reflects size, as is normal for biometric studies (e.g.
Sneath & Sokal, 1973). This ordination is shown in Figure 1, overlain with three
different sets of symbols (Fig. 1A-C).Figure 1A shows the field-recorded species
identities. Dacglorhiza incarnata forms one cluster at the negative end of the first axis
(indicating diminutive stature), whereas D. jkhsii and D. praetermissa occupy the
positive (taller) end of the first axis. The second axis separates D.fuchsii from D.
-2
-1
0
1
First principal components axis
y
2.
t
2
1-
I:
2
2
E5
L
3
Species
‘ - D purpurella
4
2
D praetermzssa 0 D. fuchszz
D zrzcarnata
t ?-hybrid
.*
0-
-1-
a
3
c -2-
$
e
1
-3
l
-
-3
I
-2
I
-1
0
1
First principal components axis
I
2
Characteristics
Lips without lines 4 Red flowers
+ None of the above
0 Spotted leaves
u
-3
-2
-1
0
1
First principal components axis
2
3
Hybrid locality
Figure 1. Thc first t\vo ascs of a Principal Components Analysis applied to thr entire dataset. This
sainr ,vaph is presented with thrcr diffcreiit labelling systcms: Labels indicate (A) provisional species
identities; (Bj presence/absrnce of tasoiiomically si<gnificantcharacteristics; (C) the substratr identity
of hyhrid plants.
395
6+
+
t
@
v
lot0
I"- l
0
h
20
I
40
I
60
80
First DCA axis
v D.purpurella
4 D. incarnata
Species
D. praetermissa
0D. fuchsii
+ ?-hybrid
Figure 2. The first two axes of a Detre'nded Correspondence Analysis ordination of the entire dataset,
with provisional species identities indicated.
praetermissa; D. fuchsii clusters at the negative end of the second axis, whereas D.
praetermissa occurs at the positive end. Figure 1B shows the presence of three
taxonomically useful characteristics (spotting on the leaves, a lack of lines on the
lip, and a red ground colour). This shows that, as a first approximation, leaf spotting
corresponds to D.fuchsii, unlined lip to D. praetermissa and red flowers to D.incarnata
subsp. coccinea. Leaf spotting was the weakest of these taxonomic indicators, with
spotted-leaved plants occurring throughout most of the ordination space. Figure 1C
shows the same diagram overlain with the distribution of putative hybrids. One
exceptional individual from the Leblanc site plots clearly with D. &chsii but was
recorded as a hybrid (shown as the black dot in the lower right-hand corner). This
plant was identical to D.fuchsii in all respects except the flower pattern which was
hyperchromic. The PFA hybrids show a weak tendency to occur in two clusters
(upper left of the figure between D. praetermissa and D. incarnata, and central right,
between D. praetermissa and D. &chsii), but the Leblanc plants exhibit no apparent
pattern.
The data were also ordinated by Detrended Correspondence Analysis. Factor
loadings are given in Table 7. The first two DCA axes (Fig. 2) show a similar pattern
to PCA. Again, the data points approximate to a triangle, with the positive end of
the first axis corresponding to D. incarnata while the negative end contains D. fuchsii
and D. praetermissa. Unlike PCA, there is no clear separation of D. fuchsii and D.
praetermissa on the second axis. Parnell & Waldren (1996) warned that DCA is
unstable when applied to bistate variables (such as leaf spotting, which was scored
as 0 or 1 in this study), and suggested re-coding such variables to 0 or n where n is
the total number of variables. In this case re-coding was found to have no effect on
the ordination.
Data were subjected to Cluster Analysis using Ward's method, nearest neighbour,
furthest neighbour and UPGMA (Unweighted Pair-Groups Method, using arithmetic
P. J. A. SHAII'
+
o+
9
+
$+*+
,
++++
+
First discriminant function
4
I
-
8
D. incarnata
I
I
-
6
4
D. praetermissa
I
10
-
2
0
U
2
4
I
6
8
First discriminant function
Figure 3. Discriminant Function Analysis plot of biometric data for species plus hybrids from PFA
sittx This graph is shown twice with two different labelling systems. Labels indicate (A) provisional
species identities; (B) presence/absence of leaf spotting. Note that the 'D.
fuduii' cluster is defined
solel!- hy leafspotting.
averages) algorithms applied to a matrix of Euclidean distances. The results are not
displaved, since the dendrograms showed poor resolution ( judged by the field-based
species identifications) and differed Miidely in their structure. Additionally, there are
practical difficulties in presenting legible dendrograms containing over 100 individuals
(Gauch, 1982).
Discriminant Function Analysis was applied using plants identified in the field to
named species as targets. This analysis explicitly assumes that the target individuals
ha1.e been correctly identified (Kent & Coker, 1992), and uses their hiometric
properties to allocate any remaining unclassified ('hybrid') individuals into the prespecified target groups. Consequently, this technique is less natural and more prone
MORPHOMETRICS OF DAC'TYLORHIZA
397
61
+
++
+
+@
00
o+ 0
aoB0'0
8 0
I
"
-
6
+
4
+
I
2
4
4
I
I
2
4
First discriminant function
-
+
+
1
4
&
++ ++ ++
++++
0
I
6
8
Species
D. incarnata
0 D. fuchsii
0 D. praetermissa
+ ?-hybrid
4
Fi<gure4. The Discriminant Function Analysis shown in Figure 3, repeated after excluding leaf spotting
from the dataset. Labels indicate species identities.
to observer error than the previous analyses, but is a logical approach to the
classification of the uncertaidhybrid plants. Separate analyses were performed for
the PFA sites and the Leblanc site, since they contained different numbers of species
(three and four respectively). Factor loadings are given in Table 7.
The first discriminant analysis for the PFA sites incorporated all biometric variables
except hyperchromism (which was excluded as it was only recorded on 'hybrid'
individuals), and produced two distinct clusters, one corresponding to D. j k h s i i and
the other containing a continuum from D. incarnata to D. praetemissa (Fig. 3A).
However, this result proved to be highly misleading when the diagram was reinterpreted in terms of the presence/absence of leaf spots (Fig. 3B). The D.fuchsii
cluster was uniquely defined by the presence of leaf spotting, and the other cluster
by its absence. This result seems overly simplistic, and contradicts the wide spread
of leaf-marked individuals within the PCA ordination (Fig. 1B). It arises as an
artefact of the discriminant analysis: almost all definite D.fuchsli had spotted leaves,
whereas none were recorded on other species on PFA, so spotted leaves were
disproportionately represented on the first discriminant axis.
The discriminant functions for the PFA sites were re-calculated after exclusion of
leaf spots, producing a very different ordination (Fig. 4). This shows a trianplar
pattern, with each corner corresponding to a named species and part of the
intervening space being occupied by hybrid plants. The hybrids occupied two distinct
regions of the ordination space: midway between D. praetemzissa and D. incarnata,
and intermediate between D. praetermissa and D. fuchsii. There were no points in the
space between B. fuchsii and D. incarnata suggesting that little if any hybridization
occurred between these two species.
Discriminant function analysis was applied separately to data from the Leblanc
site, again with leaf spotting excluded (Fig. 5). This generated one well-separated
cluster corresponding to D. incarnata; all other plants occupied an elongated cluster
L
V
4 4
*
444
r
+
+
a
*+ a-
-12
- 10
,
20
Species
I
~
1
0
10
First discriminant function
3 D.purpurella
4
D Lncarnata
D.praetermissa
0 D fuchsri
f
?-hybrid
Figtirc 5. Ilisc riiiiiiiaiit Function .\rial) sit plot of orchids from the Leblanc site (after ruclusion of leaf
spc~ttiiigj.slio\\ ing yx%cic\idrntitirs.
running from D. praeterinzs~uto D. purpuwlla. The few plants of D.praefermwa were
kvell separated from the remainder, but there was overlap between D.3ch~ii,D.
piqburdu and the hybrids.
The abo\,e analyses produce a concise description of variation within a group of
plants prone to hybridization. -4s such, it should be cxpected that there will be no
unique correct answer, and the criteria for an acceptable set of results must include
~ k g a n c eand user satisfaction (Goodall, 1978).
The ohsewation that univariatc analyses found ‘textbook’ differences between
hpccics is trivial, since the field species identifications were based on cxpected
combinations of characwrs. Of more interest are thc significant differences between
planti from PF.4 and Leblanc sites (Tahlc 6), which show plants from the Leblaric
sitc to be generally smaller and \\ith feuer flowers. This probably reflects the drier
ndture of the Leblanc site (20 I n above local nater level, compared with PFA lagoons
at. or just above. the Lvater table). Ilarploihzza furhsii and D. pruetermwu also had
flatter floltcrs at the Leblanc site. although the cause of this is unclear.
hlulti\.ariate techniques are confimed as powerful tools for obtaining an objective
o\en-iew of morphometric datasets, and for examining the extent to which preconceived species definitions are 1,alid. PCA is the most mathematically natural
ordination technique, so that the PCA ordination diagram can be used as an
ohjccti\ e summar) description of the results. This diagram (Fig. 1A-C) approximates
t o a triangle, with the corners corresponding to D.irzcarnata, D.praeternzzssa and D.
j k h > ~ zLluc!ylorhi&
.
pzirpurella occupies a cluster midway between D. praetemxtu and
MORPHOMETRTCS OI' DAC'T1LUKHIGl
399
D. incarnata, and hybrids are scattered unevenly throughout the ordination space.
This result is an unbiased description of patterns within the data and (unlike the
univariate analyses) confirms that accepted species descriptions correspond to extremes of variation within the Dacplorhiza colonies. It is possible to use positions
within this ordination diagram to estimate the parentage of hybrid individuals,
although this approach requires caution (Wilson, 1992). For PFA sites the hybrid
plants appear to occur in two loose clusters: one lies between D. jkchsii and D.
praetermissa, the other between D. praetermissa and D. incarnata. This pattern suggests
that the hybrids recorded were mainly D. praetermissa x D. fuchsii (0.x grandis (Druce)
P.F. Hunt) and D. praetemissa x D. incarnata (0.x wintoni (A. Camus) P.F. Hunt). No
clustering of hybrid plants was evident on the Leblanc site. On both sites plants
intermediate between D.fuchsii and D. incarnata were conspicuously lacking, in
agreement with the observation by Hedrtn (1996a) that these two species appear
to be genetically isolated. The results of DCA ordination generally mirrored those
of PCA, but the separation was poorer than PCA, with only D. incarnata showing
cohesion.
The failure of cluster analyses to produce a coherent or consistent classification
of the data is disappointing but not surprising. Cluster analysis is notorious for
producing information of dubious quality (Cormack, 1971) and since it seeks clear
divisions within dataspace it is ill-suited to partitioning data such as the results
presented here which approximate to a smooth multivariate continuum (Gauch,
1982).
Discriminant Function Analysis is a logical approach to the problem of classifying
individuals from a mixed population, but its results here highlighted the importance
of checking that the results of a multivariate analysis are consistent with user
experience. When this analysis was applied to orchids from PFA sites it effectively
generated an unbreakable rule that any plant with leaf spots was D.$chsii (Fig. 3B).
This simplistic result did not match field notes, nor was it compatible with the PCA
ordination. Excluding leaf spotting from the discriminant functions gave a result
that coincided more closely with PCA, namely that hybrids at PFA sites were
intermediate between either D. fuchsii and D. praetermissa or D. praetermissa and D.
incarnata. Discriminant Function Analysis, when applied to plants from the Leblanc
site, separated out D. incarnata and D. praetermissa, but left D. fuchsii, D. purjwella and
hybrids in a diffuse cluster.
Thus, two lines of numerical analysis (PCA and Discriminant Function) concur
in suggesting that introgression has produced a more even filling of multivariate
space on the Leblanc site than on PFA substrata. This may reflect the presence of
a fourth species on the Leblanc site, combined with the substantially greater age of
the habitat (c. 100 years, compared with c. 30 years for the PFA sites). Both factors
allowed greater frequency of hybridization on Leblanc. Anderson (1995) reported
greater levels of morphological diversity among Dacplorhiza traunsteineri in older
populations, although in her example population ages were estimated (from geological
factors) in thousands of years rather than decades.
It is unlikely that the taxonomic problems of this group can be solved by
morphometric techniques alone. Future work should aim to integrate morphometric
measurements with chromosomal, protein and DNA-based techniques, to explore
whether the phenotypic variability of these plants is matched by an equivalent level
of genetic variability.
?'he author is grateful to Judy Xdams (Lee Valley Park Authority), Ian Fenton
(Sational Pouer Drax). M'es Halton (hloses Gate Country Park), Alison John
iC;round\\ ork Erewash) and Dennis Vickers (London Wildlife Trust) for site access
m d hclpfiil discussions, and to the Roehampton Institute London for the granting
of \tud! leave. Erica hfcAlister and Vince Gardiner commented on drafts of this
inan uwript .
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