Genetic divergence between branched and ...

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GENETIC DIVERGENCE BETWEEN BRANCHED AND UNBRANCHED FORMS OF THE THECATE HYDROID AGLAOPHENIA PLUMA J.P. THORPE*, J.S. RYLAND*, P.F.S. CORNELIUS* AND J.A. BEARDMORE+ *Department of Environmental and Evolutionary Biology, University of Liverpool, Port Erin Marine Laboratory, Port Erin, Isle of Man. •Marine, Environmental and Evolutionary Research Group, School of Biological Sciences, University of Wales, Swansea, Singleton Park, Swansea, SA2 8PP. *Department of Zoology, Natural History Museum, Cromwell Road, London, SW7 5BD

Samples of branched and unbranched forms of the marine thecate hydroid Aglaophenia pluma (L.) (Hydrozoa, Plumulariidae) were collected from the western and eastern sides respectively of Carmarthen Bay (South Wales). Tissue from various colonies of each type was cleaned of epifauna and homogenized for horizontal starch gel electrophoresis. Useful results were obtained for 14 enzyme loci coding for a total of 11 different enzymes. Eleven of the 14 loci showed no common allele between the branched and unbranched forms. Nei's genetic identity was estimated as 018 and genetic distance as 1-7. Such a high level of genetic differentiation between morphotypes provides strong evidence that the populations are reproductively isolated and should be regarded as distinct species. It is suggested that the branched form may be Aglaophenia parvula Bale, a species until recently better known from the southern hemisphere. Morphological distinctions between the two species are discussed.

INTRODUCTION In both intertidal and subtidal areas marine hydroids are abundant, and several species, along with various bryozoans, sponges and other sessile invertebrates, are important competitors for space on hard substrata. Several hydroids are also major epiphytes of various marine algae. Most thecate hydroids of the genus Aglaophenia Lamouroux (Plumulariidae) form short plumose or feather-shaped growths up to a few centimetres in height. Some others form tall, branched cormi comprising several plumes. However, even within relatively well-worked European waters the taxonomic relationship between single-plumed and multi-plumed morphotypes is often unclear. Thus it has been suggested that the singleplumed A. lophocarpa might merely be the young of the multi-plumed A. acacia, although this view is now discounted (Svoboda & Cornelius, 1991) it nevertheless highlights the problem. Again, in some Mediterranean populations of A. tubiformis, it has been suggested that branched colonies are produced at sea temperatures below 16°C, in winter, whereas summer growths are said to remain unbranched (Svoboda & Cornelius, 1991). A third such problem concerns the commonly reported, but taxonomically confusing, A. pluma (L.). In this species, at least in British waters, upright stems usually form single

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plumes, but occasional colonies are branched and therefore multi-plumed. Despite the lack of any conspicuous difference in fine structure, some authors {e.g. Kirchenpauer, 1872; Teissier, 1965) have regarded branched forms of A. pluma as a distinct species, although most {e.g. Johnston, 1838,1847; Hincks, 1868; Millard, 1957) have considered branched and unbranched forms to be conspecific. Unbranched colonies of Aglaophenia pluma are predominant around the British Isles and apparently occur both on brown algae (most commonly Halidrys siliquosa (L.) Lyngb.) and on hard substrata. Each upright stem contains numerous individuals which are asexually produced and thus are from a single clone. Clones can also expand horizontally by means of stolons which spread runner-like across the substratum and give rise to further upright plumes. The stolons are very inconspicuous, and under natural conditions it is very difficult to tell whether adjacent plumes are from the same colony. The presence of reproductive structures indicates that the species reproduces sexually, mainly in summer (Svoboda & Cornelius, 1991 and references therein). The branched form is thought to occur from southern Britain to the western Mediterranean (Svoboda & Cornelius, 1991). The material collected for the present study extends the recorded distribution of the branched form to south-west Wales. Warm summer sea temperatures have been suggested as promoting the growth of branched colonies, on the assumption that they are conspecific with A. pluma. However, little winter-collected material has been available for study (Svoboda & Cornelius, 1991); and the preponderance of unbranched A. pluma during summer in north-west European waters suggests that temperature might not be the only factor. Although probably not rare, branched colonies are not easily located. Thus the finding of an intertidal source of limited material in south-west Wales provided an opportunity to compare that form with the more common single-plumed form. The most instructive method for the investigation of the relationship between these two growth forms of Aglaophenia pluma was considered to be enzyme electrophoresis. This method permits the identification of the particular alleles (genes) present at a range of chosen gene loci (coding for various enzyme molecules) in each individual organism studied. It is thus a powerful tool for the identification of separate gene pools, and is of considerable value in distinguishing species and for other systematic studies (e.g. Avise, 1974; Ayala, 1983). Earlier electrophoretic studies of hydroids are reviewed by Ostman (1987). Among other cnidarians the technique has provided evidence of speciation in various sea anemones (e.g. Carter & Thorpe, 1981; Haylor et ah, 1984; Shaw et ah, 1987) and has also been used for similar work in several other marine invertebrate phyla (e.g. Thorpe et ah, 1978a; Sole-Cava & Thorpe, 1986). MATERIALS AND METHODS Samples of unbranched Aglaophenia pluma were collected from low-shore gullies at Crabart (Worm's Head Causeway, Gower Peninsula, South Wales) on 14 July 1987. Branched colonies were also collected intertidally, from a large low-shore rock pool near Lydstep Head (South Pembrokeshire, West Wales). Samples were identified as A. pluma from the detailed descriptions of Svoboda & Cornelius (1991). Only four species of the

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genus are considered to occur in Britain (Svoboda & Cornelius, 1991). Unexpected difficulties were encountered in obtaining samples from a number of different clones. It was intended to eliminate duplication of clonal samples by using electrophoresis to identify colonies of different genotype (a method previously used in sea anemones; Sole-Cava & Thorpe, 1992). However, this proved impossible in the Crabart samples since none of the 14 loci studied was polymorphic, and thus there was no scope for these to be used to identify clones. Therefore many potentially different samples were discarded because it was impossible to tell whether they were actually different. The only samples used were one from each of the five different areas collected at Crabart. At Lydstep all the samples found were from a single rock pool. A single variable locus enabled three different genotypes to be identified and thus at least three different clones were present. Since all samples were from the same general area only a single sample from each of the three clones was used. All the samples were maintained alive in the laboratory until required for electrophoresis. Before use they were carefully cleaned to remove caprellids and other associated fauna; a laborious procedure taking up to about half an hour per colony. Electrophoresis was carried out using standard horizontal starch gel techniques (e.g. Harris & Hopkinson, 1978; Richardson et al., 1986). Gels used 12-5% starch (Sigma Chemical Table 1. Aglaophenia pluma. Alkie frequencies at 14 enzyme loci for samples of unbranched material from Crabart (A) and branched material from Lydstep (B) Enzyme

Locus

Allele

A

Superoxide dismutase (E.C. 1.15.1.1)

Sod-1

1 2 1 2 1 2 1 2 1 2 1

1.00 0.00 1.00 0.00 0.00 1.00 1.00 0.00 1.00 0.00 1.00 0.00 0.00 1.00 0.00 0.00 1.00 0.00 1.00 0.00 1.00 1.00 1.00 0.00 0.00 1.00 1.00

Sod-2 Peptidase(E.C.3.4.11-.13-)

Pep-1 Pep-2

Phosphoglucose isomerase (E.C. 5.3.1.9)

Pgi

Phosphoglucomutase (E.C. 5.4.2.2)

Pgm

Leucine aminopeptidase (E.C. 3.4.11.1)

Lap-1

2 1

2 Lap-2

1

2 Sorbitol dehydrogenase (E.C. 1.15.1.1)

Sdh

Xanthine dehydrogenase (E.C. 1.2.3.2) Malate dehydrogenase (E.C. 1.1.1.37) 6-phosphogluconate dehydrogenase (E.C. 1.1.1.44)

Xdh Mdh

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49)

G-6-pdh

Isocitrate dehydrogenase (E.C. 1.1.1.42)

Pgd

Idh

3 4 1 2 1 1 1 2 1 2 1

B 0.00 1.00 0.00 1.00 1.00 0.00 0.00 1.00 0.00 1.00 0.00 1.00 1.00 0.00 0.17 0.17 0.50 0.17 0.00 1.00 1.00 1.00 0.00 1.00 1.00 0.00 0.00

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Company, Poole, Dorset, England). Following trials using various buffers a continuous tris-citrate system pH 8-0 from Ward & Beardmore (1977) was used. Tissue samples were homogenized in one drop of gel buffer. Staining methods followed those of Shaw & Prasad (1970) and Harris & Hopkinson (1978). For peptidase the stain used the dipeptides, leucyl glycine, glycyl glycine, glycyl valine and phenylalanyl tyrosine, and the tripeptide, leucyl glycyl glycine. RESULTS Informative results were obtained for a total of 14 enzyme loci coding for a total of 11 different enzymes. Allele frequencies at these loci for both branched and unbranched Aglaophenia pluma are shown in Table 1. Enzyme nomenclature follows that of Harris & Hopkinson (1978). Of the 14 loci studied only two (Xdh and Mdh) showed no differences between the branched and unbranched forms of Aglaophenia pluma. At 11 of the 14 loci studied no allele was found to be common to the two populations. Consequently each of these loci showed significant (P