Please find below the final report for Wildlife Grant number 13487 – Diversity in Irish Polyplacophora




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Please find below the final report for Wildlife Grant number 13487 – Diversity in Irish Polyplacophora.  There are two files – this file, containing the report and appendices .

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 Ter-minatorv3.0 (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 majority-rule (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 under-sampled. 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 most-derived 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.





Character definition

States

1

animal body shape

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

loci sequenced

(1) Leptochiton asellus

16S, 18S, 28S

(5) Ischnochiton albus

18S

(6) Callochiton septemvalvis

18S, 28S

(7) Lepidochitona cinereus

16S, 18S

(10) Acanthochitona crinita

16S, 18S, 28S

(12) Acanthochitona fascicularis

28S




A






B



A




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.


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