Please find below the final report for Wildlife Grant number 13487 – Diversity in
Irish Polyplacophora. There are two files – this file, containing the report
<sigwart_chiton.doc> and appendices <sigwart_chiton_appendix.doc>.
Printed copies have also been sent to you by post.
Work on this project is now complete. Funding is now requested for remuneration for
coverage of the project costs, including travel and laboratory services, for the agreed
award—a total of € 6000.
n.b. Funding for field equipment, field accommodation, and field supplies including
equipment for specimen preservation were provided by UCD Zoology departmental research
funds, the National Museum of Ireland (for deposited voucher specimens) and/or by JDS.
Library services were funded by JDS.
BUDGET
Travel
Background research travel: Ulster Museum
(one trip, Dublin to Belfast)
€ 200
Travel to field sites May-June 2004
(two trips to each of three sites)
€ 1000
Laboratory services
Primer determination, sequencing and enhancement
(for 16 specimens @ € 300 ea. incl. VAT) € 4800
REQUESTED AMOUNT
€ 6000
Diversity in Irish Polyplacophora
Heritage Council Wildlife Grant Number 13487
Julia D. Sigwart
November 2004
Summary
The polyplacophorans or ‘chitons’ are distinctive marine invertebrates found on rocky
shores around Ireland. These ‘primitive’ molluscs are algal grazers, identified by eight shell
plates or valves, and spend their whole lives clinging to rocks in the intertidal. However, very
little is known about the species diversity or evolutionary relationships among Irish chitons, or
even the class in general. Some baseline data is available for the distribution of these animals
around Ireland, but their reactions to potential disturbances such as pollution or climate change
are completely unknown. This project contributes new information from original field
observations and from DNA-sequencing. The resulting analysis has produced the first-ever
chiton ‘family tree’ for Irish polyplacophorans, and adds new understanding to the diversity of
this enigmatic group. This project has also produced the first-ever Checklist of Irish Chitons,
including details of the names and distributions of the twleve (12) Irish species. By studying
polyplacophorans, we can learn not only about the diversity of their group, but also about the
origins and continuing evolution of rocky intertidal ecosystems in Ireland.
Introduction
The chitons (class Polyplacophora), or ‘coat-of-mail shells’ (cióton máille), are some of the
most enigmatic of the familiar marine invertebrates from rocky intertidal ecosystems. Chitons
form a distinctive molluscan clade whose members normally have eight shell plates (valves) as
adults. These animals are found in oceans all over the world, primarily in the intertidal zone of
exposed rocky shores. The shell plates of chitons, in a row on their dorsum, are the most
distinctive characteristic of the group and the often their only visible feature. Chitons have no
real eyes—only a foot, and a mouth, under their shells—but most species have photosensitive
spots on the edges of their shells, which provide some insights for them to the outside world.
The chitons spend their whole lives in the intertidal zone, where they cling to rocks with a strong
foot and feed on encrusting algae. Most species of chitons measure two to three centimetres in
length, although there are several found in the West of Ireland which can be almost 8 cm long as
adults. Because the evolutionary lineage of chitons is thought to have diverged from other extant
mollusc groups early-on, and because the overall appearance and habits of chitons have
apparently not changed much over their half a billion year history, this “living fossil” group is of
considerable interest to the study of early molluscan evolution. However, the bulk of
polyplacophoran scientific literature does not consider the place of the species we find on Irish
shores.
Here, for the first time, I present a checklist of Irish Polyplacophora. I can confirm records of a
total of twelve (12) species on Irish shores. This is a new and exciting result, to finally know
how many species of chitons there really are on Irish shores. The present project is fundamental
to forwarding the larger research goals of studying chiton evolution, while answering interesting
questions about the actual species diversity of Irish chitons. Analysis of this type has never
before been attempted on the Irish malacofauna, but it is also important to bear in mind the
large-scale need for detailed base-line information on near-shore fauna. Shallow marine
habitats are most susceptible to the influences of global warming and corresponding increases in
sea surface temperature. Because chitons seem to represent a more “primitive” sort of life form
than many more familiar molluscs, like the octopus, they can help us understand the
development and evolution of this large and complex group of organisms. Through field
collection and analysis, this project presents the first-ever ‘family tree’ for Irish
polyplacophorans. The central aim of this project has been to address the question of how many
species of polyplacophoran molluscs are present in Ireland, and what can be learned about their
evolutionary relationships. Through studying the development and relationships of these
creatures, we gain in our understanding of the origins and continuing evolution of Ireland’s
natural heritage.
Methods
A great wealth of material about historic and present distributions of the Irish fauna are held
in the research collections of the National Museum of Ireland (Natural History, Dublin) and the
Ulster Museum (Belfast). However, because these records have been accumulated continuously
for 200 years, the majority are not accessible or even summarised in any digital format. Thus,
extracting information is time- and labour –intensive, but ultimately extremely rewarding.
In addition to direct specimen evidence, I relied on scientific literature and historical and
contemporary field guides to seashells that made specific reference to chitons. Many older field
guides (ca. 1950 and earlier) are aimed at shell collecting enthusiasts, and offer species-level
diagnoses for common shore animals. More contemporary field guides, which are designed
more for use observing creatures in their own habitats are often less specific for animals that are
difficult to identify, such as chitons. Records were only included in the summary of distribution
data if multiple Irish records were found for a species, and if a relatively specific locality
information could be determined.
To study evolutionary relationships, I analysed sequence data for six species – half of the
Irish diversity. Comparative sequences were also acquired from colleagues at Harvard
University (Cambridge, USA) for several additional species to be included for comparative
purposes. For Irish samples, total DNA was extracted from a small tissue sample of each
individual, from the body wall, foot or gonads, using the DNeasy Tissue Kitfrom QIAGEN ©
and the protocol provided by the manufacturer. The purified total DNA was used as template for
amplification of a portion for each of the 16S, 18S, and 28S rRNA using the polymerase chain
reaction (PCR). The complete rRNA (ca. 1.8 kb) was amplified in overlapping fragments, using
primer sequences provided by colleagues in Harvard University. The PCR reactions were
carried out using a GeneAmp PCR System 9700 thermal cycler, and involved an initial
denaturation step (5 min at 95 °C) followed by 35 cycles including denaturation at 95 °C for30 s,
annealing (ranging from 45 to 49 °C) for 30 s, and extension at 72 °C for 1 min, with a final
extension step at 72 °C for 1 min. The double-stranded PCR products were verified by agarose
gel electrophoresis (1% agarose), and purified using GENECLEAN II Kit (BIO 101). The
purified PCR products were sequenced directly; each sequence reaction of a total volume of 10
µL included 2 µL of the PCR product, irrespective of PCR yield, 1 µM of one of the PCR primer
pairs, 2 µL of halfTERM Dye Terminator Reagent (Genpak), and 2 µL of ABI BigDye TM Terminatorv3.0 (Applied Biosystems). The sequence reactions, performed using the thermal cycler
described above, involved an initial denaturation step for 3 min at95 °C, and 25 cycles (95 °C for
10 s, 50 °C for 5 s, 60 °C for 4 min). The BigDye-labelled PCR products were cleaned with
AGTC ® Gel Filtration Cartridges (Edge BioSystems). The sequence reaction products were
then analysed using an ABI Prism 3100 Genetic Analyzer.
For morphological analysis, I studied the formation of ten (10) cladistic characters across all
Irish species as well as # additional taxa used as outgroups for molecular analysis. To determine
correlation between morphological and molecular signals, the two datasets were analysed
separated and compared. Cladistic analyses were performed used the standard software
Phylogenetic Analysis Using Parsimony (PAUP) heuristic search. Resultant trees were
combined as 50% majority-rule consensus agreement.
Results
Based on field work, biological records, and literature, I have complies a Checklist of the
Irish Chitons including twelve (12) species found on the island of Ireland (see Appendix 1).
There is one additional species which may be present: Chiton olivaceus; however, I have not
been able to satisfactorily validate this record. The scientific names of the Irish species are from
a variety of sources and have been continuously studied by various biologists from their
populations throughout Europe (see Box 1). The checklist includes complete synonymy of other
scientific names that have been used for Irish species. All field records and literature references
have been compiled to create rough distribution maps for the Irish chiton fauna (see Appendix
2).
Of the 12 species found in Ireland, four (4) are in the suborder Leptochitonina, considered to
be the most primitive polyplacophorans. The remainder are divided between the five (5) species
in the family Ischnochitonidae three (3) species in the Acanthochitonidae. In traditional
classification, these three groups are arranged in ‘ascending’ evolutionary order, with
acanthochitonids representing the crown group of Polyplacophora, and with the base of living
chitons in a clade comprising Leptochitonidae and Hanleyidae within the Leptochitonina. This
classical interpretation differs substantially from the results obtained through rigorous analysis.
Molecular sequences yielded varying lengths of intact rRNA at different loci. The largest
partition was 1840 base-pairs (bp) on 18S rRNA; 28S yielded 1044bp and 16S yielded 544bp
over all taxa. Of the six Irish species sampled, not all loci were available for all species (see
Table 1). Overlap was deemed sufficient to combine sequences linearly, in combination with
additional overlapping sequences from non-Irish taxa (see Appendix 3A).
Cladistic analysis of molecular data resulted in 5523 most-parsimonious trees (MPTs).
Strict consensus produced a polytomy with no separation of the defined outgroups. A majorityrule (50%) consensus retained the topologies of most MPTs (see Figure 2). In this molecular
tree, taxa are divided into five clades. However, there is still no resolution of rooting by the
outgroups. Irish species divide into three clades, without any of the traditional relationships
retained. In particular, the two Acanthochitona species are divided, with A. fascicularis set
entirely on its own.
Separately, ten (10) morphological characters were defined from external features (see Box
2). Codes for all characters across all species were included in a cladistic data matrix (see
Appendix 3B). Analysis resulted in 627 MPTs. Strict consensus again produced an unresolved
polytomy. The structure retained by the majority-rule (50%) of all MPTs indicates considerably
more structure than the molecular data, and with different patterns emerging (see Figure 3).
Irish species are found in two clades. All three of the Acanthochitona spp. group together as
the crown group of the more basal clade. Following classical taxonomy, the basal clade also
contains the Leptochitonina in one leptochitonid species and its sister-group, represented by
Hanleya. The second, crown clade contains all of the ischnochitonids, but also includes L.
asellus, another ‘primitive’ leptochitonid.
Discussion
Polyplacophorans are a diverse and important group of molluscs that are often overlooked
because their identification is comparatively more difficult than other groups. The Checklist of
Irish Chitons (included in Appendix 1) is a useful tool for the future investigation of a very
difficult and under-studied group of marine invertebrates. The twelve (12) species in Ireland
represent a great deal more biodiversity than was previously recognised.
Chitons are found in a wide range of depths in the marine environment, from the low
intertidal (e.g. Tonicella) to abyssal depths (e.g. Leptochiton). Also, in all ecosystems, chitons
tend to be found in low densities. Many species included on the Checklist are clearly undersampled. Even with comprehensive data available from surveys like the BioMAR project in the
1990s, there are still significant gaps in our knowledge.
Distribution of Irish species is not well understood, and our current knowledge appears to
primarily reflect patterns of intensive collecting (see Appendix 2). The majority of records
come from Galway, Cork, Dublin, and popular localities in Northern Ireland (e.g. Mulroy Bay,
Strangford Lough). Additional survey work, with reliable species-level identification, will be
needed to fill the gaps around the coast of Ireland.
Importantly, the present morphological analysis also fails to support the traditional structural
hypothesis for chiton evolution. Acanthochitona and its allies are considered to be the mostderived polyplacophorans. However, these results clearly imply they share a closer common
ancestor with the ‘primitive’ Leptochiton. This is a very intriguing possibility.
It is also interesting to note that the two Tonicella spp. are not grouped together, and that T.
rubra is more closely allied with Lepidochitona. Tonicella spp. in particular are morphologically
very diverse. The same genus in Pacific waters has recently been recognised to contain a large
and complex suite of species, many of which have deceptively similar shells. The same may
prove true in the North Atlantic.
It is now generally accepted by evolutionary biologists that the ‘classical’ interpretation of
chiton relationships is very inadequate. The present study, although preliminary, highlights the
deep problems in rationalising molecular signals with taxonomic hypotheses. These results
illustrate that we are far from understanding the radiation of modern chitons in the North
Atlantic, or indeed anywhere. The molecular analysis of Irish chitons appears to put the species
in an arrangement that is close to random. However, this family tree may very well represent the
true history of chiton evolution, much more so than previous superficial interpretation. But as a
hypothesis for polyplacophoran systematics, it is too dramatic a departure to be accepted without
more detailed analysis. More taxa must be included from the global chiton fauna, to determine if
these species share closer common ancestors with cousins that are now separated by continents.
We know that marine faunas are sensitive environmental indicators for impacts of pollution,
habitat destruction, invasive species, and over-exploitation. In particular, ecosystems in shallow
seas (such as most of this material represents) may be especially sensitive to the effects of global
warming and consequent increases in ocean temperature. However, studies of biodiversity often
suffer from a lack of reliable baseline data with which to compare our current findings. Museum
collections are physical databases of our planet's biota. Recent studies (2000) have found that
recent museum collections independently recover 80 per cent of species found in intensive
nearshore surveys.
Additional work by many ecologists indicates that nearshore
macroinvertebrates such as molluscs are highly sensitive to changes in their marine
environments. Specimen databases in museums are one tool that allows us to analyse in detail
patterns of biodiversity in space and time, in response to changing conditions. More work on
Irish species is needed to facilitate ecologists and all others interested in natural heritage, who
may not have previously appreciated that such unassuming creatures can contain such complex
questions of evolutionary biology.
Box 1. Publications of the original taxonomic descriptions of chitons found in Ireland (in
alphabetical order).
Brown T., 1827 :
Illustrations of the recent Conchology of Great Britain and Ireland. W.H. & D. Lizars,
Edinburgh pp. XIV + 125 + 52 pl
Acanthochitona discrepans
Fabricius O., 1780 :
Fauna Groenlandica systematice sistens animalis Groenlandiae occidentalis hactenus indagata.
Hafniae et Lipsiae, J.G. Rothe pp. XVI + 452 + 1 pl
Tonicella marmorea
Gmelin, J. F., 1791 :
Carli Linnaei systema Naturae per regna tria naturae. Editio decimatertia, aucta, reformata,
Vermes Testacea. Leipzig [Lipsiae] 1 (6): 3021-3910 [molluschi]. [pre 14 maggio 1791: vedi
Hopkinson, 1908]
Leptochiton asellus
Jeffreys J. G., 1880 :
On a new species of Chiton lately found on the British coast Annals and Magazine of Natural
History (5) 6: 33-35
Leptochiton scabridus
Kaas P., 1985 [revised] :
The genus Acanthochitona Gray, 1821 (Mollusca, Polyplacophora) in the north-eastern Atlantic
Ocean and in the Mediterranean Sea, with designation of neotypes of A. fascicularis (L. 1767)
and of A. crinita (Pennant, 1777) Bulletin du Muséum National d'Histoire Naturelle (section A
Zoologie) (4) 7 (3): 579-609
Acanthochitona crinita; Acanthochitona fascicularis
Linné C. von, 1766-1767 :
Systema Naturae. Editio duodecima. 1. Regnum Animale. 1 & 2 Holmiae pp. 1-532 [1766] pp.
533-1327 [1767]
Ischnochiton albus; Lepidochitona cinerea; Tonicella rubra
Montagu G., 1803 :
Testacea Britannica, or natural history of British shells, marine, land and the fresh-water,
including the most minute: systematically arranged and embellished with figures Romsey.
London pp. XXXVII + 606 + 16 pl. (dicembre)
Callochiton septemvalvis
Thorpe C., 1844 :
British marine conchology. Lumley, London LX + 267 p.
Hanleya hanleyi
Box 2. Numerical encoding for anatomical characters used in cladistic analysis.
1
Character definition
animal body shape
States
0: ovate
1 : elongate
2
elevation (height / width) of intermediate plates
0 : ratio > 0.4
1 : ration ≤ 0.4
3
valves beaked
0:N
1:Y
4
apices usually worn
0:N
1:Y
5
valves keeled (‘carinate’)
0:N
1:Y
6
intermediate plates with distinct diagonal seperating
lateral areas
0:N
1:Y
7
mucro prominent
0:N
1:Y
8
girdle appearance
0 : leathery or scaley
1 : with bristles or projections
9
distinctive growth rings
0:N
1:Y
10
gill description
0 : abanal
1 : adanal
Table 1. rRNA loci included in molecular analysis of Irish polyplacophoran species.
Species
(1) Leptochiton asellus
(5) Ischnochiton albus
(6) Callochiton septemvalvis
(7) Lepidochitona cinereus
(10) Acanthochitona crinita
(12) Acanthochitona fascicularis
loci sequenced
16S, 18S, 28S
18S
18S, 28S
16S, 18S
16S, 18S, 28S
28S
A
A
B
Figure 1. Irish chitons in their natural habitat. A. Leptochiton asellus B. Tonicella marmorea.
Photos courtesy B.E. Picton, Ulster Museum.
Figure 2. Molecular reconstruction of relationships among chitons including species found in
Ireland, 50% Majority-rule consensus of 5523 trees. Grey highlighting of branches indicates
Irish native species; others are worldwide species used in analysis for comparison. Black bars on
the right-hand side indicate the five distinct groups or ‘clades’ that appear to segregate species
analysed. Outgroup placements have been removed from their positions internal to the present
clades to improve clarity.
Figure 3. Reconstruction of relationships among chitons including species found in Ireland from
morphological characters, through 50% Majority-rule consensus of 627 trees. Grey highlighting
of branches indicates Irish native species; others are worldwide species used in analysis for
comparison. Black bars on the right-hand side indicate the five distinct groups or ‘clades’ that
appear to segregate species analysed.