Defined by genitalia in the Madeiran endemic land snail genus Heterostoma

Heterostoma is a small helicid, endemic to the Madeiran Islands. It exhibits two distinct forms of genitalia: a hemiphallic form which lacks a flagellum and has a much reduced penis and epiphallus and a euphallic form with a fully developed set of male outcrossing organs. Some believe this genital variation and associated differences in shell morphology are sufficient to warrant division of the genus into two species. Others suggest this is an example of a stable, intraspecific polymorphism as seen in some other gastropod species, notably Bulinus truncatus. Resolution of this problem is essential if the evolutionary significance of linked genital and morphological variation is to be understood.

Genetic analysis of allozymes has proved inconclusive, largely through technical difficulties limiting the number of loci resolved. More recently developed techniques of molecular genetic analysis can greatly increase the number of loci available for study. They also allow for direct analysis of DNA itself, rather than its protein products. This second advantage allows analysis of areas of the genome that do not code for proteins. Regions of this non-coding DNA change at rates which are often very different from the regions involved in protein coding. Together, these advantages greatly increase the resolution of molecular techniques compared to allozyme methods and make them especially useful in the analysis of difficult phylogenies.

This study used the technique of randomly amplified polymorphic DNAs (RAPDs). Standard methods for gastropod DNA analysis were used. Samples of snails were taken from various sites on two islands in the Madeiran archipelago: Porto Santo and one of its smaller, satellite islands, Ilheu de Cima, with snails assigned to taxon by dissection of the genitalia. RAPD analysis with 10 primers gave 192 bands in 48 individuals. Pairwise comparison of bands between individuals resulted in a dissimilarity matrix which could be used to generate principal co-ordinates for each snail. Plotting the first two of these gives the results shown in Figure 1. Hemiphallics from Porto Santo clearly form a separate cluster which does not overlap with euphallics. Euphallics form a different cluster with overlap between cases from Porto Santo and Ilheu de Cima. Hemiphallics from Ilheu de Cima plot very close to the euphallic cluster but do not overlap it. These results suggest a clear genetic division between hemiphallics and euphallics, at least on Porto Santo. Calculation of Nei and Li's F statistic and averaging for cases from the same sample site allows an UPGMA dendrogram to be calculated, as shown in Figure 2. Hemiphallics and euphallics form well separated clusters, with hemiphallics from Ilheu de Cima forming an outgroup within the main hemiphallic cluster. There is no separation of euphallic samples by island.

These results suggest that hemiphallic and euphallic Heterostoma act largely as evolutionarily independent units. They may retain the ability to hybridise (as evidenced by Ilheu de Cima hemiphallics) although this would have to occur at an extremely low rate.While such an ability to hybridise strictly contravenes the biological species concept, it seems reasonable to suggest that the taxa of Heterostoma are, by and large, evolutionarily independent of each other.

I would like to thank the Malacological Society of London for financial support in the form of a Centenary Research Grant. I am also very grateful to Les Noble and Cathy Jones, University of Aberdeen, for use of facilities and for technical advice.

Paul Craze

Department of Biological Sciences

Manchester Metropolitan University