Heredity 79 (1997) 463—472 Received 20 August 1996 Biochemical systematics and evolutionary relationships in the Trichoniscus pusillus complex (Crustacea, Isopoda, Oniscidea) MARINA COBOLLI SBORDONI1, VALERIO KETMAIERff, ELVIRA DE MATTHAEIS & STEFANO TAITI Dipartimento di Scienze Ambienta/i, Università di L 'Aquila, V. Vetoio, Local/ta Coppito-67010-L 'Aqu/la, Dipartimento di Biologia An/male e dell'Uomo, Università di Roma La Sap/enza', V./e de/I'Univers/tà 32- 00185-Rome and §Centro di Studio per/a Faunistica ed Eco/ogia Tropicali CNR, V.Romana 17-50125- Florence, Italy Inorder to clarify taxonomic and phylogenetic relationships among Trichoniscus pusillus (Isopoda, Oniscidea) populations, allozyme variation was studied by means of starch gel electrophoresis. The genetic structure of several populations belonging to different subspecies (diploid bisexual, triploid parthenogenetic; epigean, troglophilic and troglobitic) was assessed by investigating 10 enzymatic systems corresponding to 15 putative loci. F-statistics and cluster- ing analysis indicated a high degree of genetic differentiation, corresponding to low levels of gene flow among populations, both epigean and hypogean, still considered to be conspecific. Estimates of divergence times calculated from genetic distance data suggest that the pattern of differentiation and the colonization of cave environments may be related to the palaeoclimatic change of the Messinian and PIio—Pleistocene glaciations. Keywords: allozymes, cave fauna, divergencetimes, genetic polymorphism, phylogeny, Tricho- niscus pusillus. Introduction accepted, is arbitrary and subjective. Thorpe (1987) stressed that most species in natural circumstances Trichoniscuspusillus Brandt, 1833 (Isopoda, Onisci- may have patterns of geographical variation. As dea) is considered to be a polytypic species, widely conventional subspecies are not natural categories, distributed in the Palaearctic region, whose popula- tions have been arranged in several subspecies. their use consequently forces noncategorical varia- These subspecies may occur at or above the soil tion into categorical classes. Several approaches surface (epigean), in caves and subterranean passa- have been proposed to obtain a more realistic defini- tion of subspecies (Böhme, 1978; Thorpe, 1987), all ges but not strictly confined to them (troglophilic), based on careful descriptions of morphological, or only in caves (obligate cavernicolous, troglobitic). ecological and interfertility parameters. On the Moreover, they differ reproductively (diploid bisex- ual vs. triploid parthenogenetic) and show a strong other hand, many systematists continue to accept homogeneity for the morphological characters tradi- conventional infraspecifIc categories as a useful tool tionally used in the systematics of this group. On the for describing patterns of variation, especially in animals with limited dispersal power and discontin- other hand, they exhibit some variability in adaptive characters related to the colonization of subter- uous distributions. This allows recognition of more ranean environments (loss of the eye, body depig- or less differentiated populations without demon- strable sexual isolation, especially when the pheno- mentation, etc.). The use of conventional subspecies is much typic differences are less than the average between debated, even though they are widely employed in recognized species in the same genus. the study of geographical variation caused by Analysis of the amount of genetic divergence ecological patterns and/or historical processes. between populations considered to belong to Böhme (1978) has pointed out that the subspecies different subspecies should be a way of elucidating concept, as defined by Mayr (1975) and generally patterns of variation and differentiation. The present paper deals with the genetic structure of several *Correspondence E-mail: [email protected] natural populations belonging to different subspecies 1997 The Genetical Society of Great Britain. 463 464 M. COBOLLI SBORDONI ETAL. of Trichoniscus pusillus. In addition, a population of CAP; Elba Is., ELB; Gorgona Is., GOR), of Tricho- Trichoniscus matulicii Verhoeff, 1901, has been niscus pusillus provisorius Racovitza, 1908, a taxon assayed to assess levels of genetic divergence among widely distributed in Europe, especially in the morphologically well-differentiated species in the southern part, and the Mediterranean (Algeria, genus Trichoniscus. Lebanon). One population is of Trichoniscus pusillus baschierii Brian, 1953, a troglobitic form endemic to Materials and methods the Punta degli Stretti cave, Monte Argentario, Grosseto, Tuscany (STR). Three populations are of Study area and collecting sites a new troglophilic taxon (Taiti & Ferrara, 1995), Twelve populations (identified by three-letter codes) belonging to the Trichoniscus pusillus complex, living were analysed (collecting sites are indicated in Fig. in some natural caves of the Tuscan Apennines 1). One population was obtained from Ulbach near (Buca presso il Trogolin dell'Orso di Vallombrosa, Stuttgart (Germany, PUS) and belongs to the Florence, VAL; Buca delle Fate di Tosi, Florence, nominal subspecies Trichoniscus pusillus pusillus, TOS; Buca delle Fate di Badia Prataglia, Arezzo, widespread in central and northern Europe. This is a FAT). A population of Trichoniscus pusillus sujensis triploid form (3n =24) characterizedby an obligate Brian, 1926, is from the type locality, Grotta Suja, apomictic parthenogenesis (Vandel, 1960). Five are Monte Fasce, Genova, Liguria (SUJ). Finally, one epigean populations, both continental (Sant'Ellero, population of Trichoniscus matulicii, a species with a near Florence, ELL) and insular (Francardo, trans-Adriatic distribution (Argano et al., 1978), Corsica, FRA; Tuscan Archipelago: Capraia Is., comes from the Punta degli Stretti cave, Monte Argentario, Grosseto, Tuscany (MAT). All samples were transported alive to the laboratory and frozen at —80°C. Electrophoreticanalysis Horizontaielectrophoresis was performed on 12 per cent starch gels with crude homogenates in Tris-HC1 0.05 M pH 7.5 from each whole specimen. Ten enzymatic proteins were assayed for genetic varia- tion: acid phosphatase (ACPH, EC 3.1.3.2), alkaline phosphatase (APH, EC 3.1.3.1), esterase (EST, EC 3.1.1.1), glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49), isocitrate dehydrogenase (IDH, EC 1.1.1.42), lactate dehydrogenase (LDH, EC 1.1.1.27), nonspecific dehydrogenase (NO-DH, EC 1.6.99.1), peptidase (PEP, EC 3.4.11.-), phosphoglucomutase (PGM, EC 5.4.2.2), phosphohexose isomerase (PHI, EC 5.3.1.9).These enzymes correspond to 15 puta- tive loci. Buffers and electrophoretic conditions were according to De Matthaeis et a!. (1983) and Cobolli Sbordoni et al. (1987). The genetic relationships between diploid popula- tions were estimated on the basis of genetic distance (D) values, calculated with Nei's method (1978) applied to the allele frequencies at the 15 common loci. To quantify the amount of genetic differentia- tion between the diploid populations and the trip- bid one, genetic similarity (S) was calculated on the basis of genotype frequencies according to Hedrick's formula (1971), because it is usually impossible to decide which of two alleles is present in double dose Fig. I Sampling localities of Trichoniscus populations. For in heterozygous triploids. Genetic relationships the population symbols see text. among all populations are represented by a dendro- The Genetical Society of Great Britain, Heredity,79, 463—472. MICROEVOLUTION IN THE T. PUSILLUS COMPLEX 465 gram plotted with the UPGMA clustering method 1; Acph-2; Aph-i; Aph-2; Est-i; Est-2; Est-3; G6pd; (Sneath & Sokal, 1973). Idh; Ldh; Pep-i; Pep-2; Pgm; Phi) were polymorphic On the basis of the values of Rogers genetic at least in one population. In several cases alterna- distance (1972), with the method of outgroup tive fixed alleles were found among populations. On rooting, a tree was drawn to estimate the phylo- the basis of genotype frequency data, the genetic genetic relationships between populations by means similarity index (S) (Hedrick, 1971) was calculated of the distance Wagner procedure (DWP) (Farris, between all study populations (Table 1), and the 1972). genetic identity and distance indexes (I; D) (Nei, The degree of genetic heterogeneity among all 1978) were employed to quantify genetic relation- populations of T pusillus was assessed using the 0 ships among diploid populations (Table 1); the index (Weir & Cockerham, 1984) as an estimator of values obtained by the two methods gave patterns of FST. An indirect estimate of gene flow is given by: differentiation of the same order of magnitude. S Nm =(1/FsT—l)/4(Wright, 1931), where N is the varied from 0.848 (VAL vs. TOS) to 0.138 (MAT vs. effective population size and m is the effective ELB), whereas I varied from 0.965 (VAL vs. TOS) migration rate. Moreover, in order to obtain a to 0.159 (MAT vs. ELB). The dendrogram of Fig. 2, detailed description of patterns of genetic hetero- constructed from the S-values, synthesizes genetic geneity and gene flow among Tuscan and Corsican relationships, showing the existence of different populations, we arranged them in five groups on the levels of genetic differentiation; the tree built basis of their geographical area: Apennine cave according to the distance Wagner procedures populations (VAL, TOS, FAT); Tuscan cave popula- (Farris, 1972), on the basis of Rogers genetic tions (STR, VAL, TOS, FAT); epigean—hypogean