The Genetics of Maintenance of an All-Male Lineage in the Squalius Alburnoides Complex

Journal of Fish Biology (2002) 60, 649–662 doi:10.1006/jfbi.2002.1883, available online at http://www.idealibrary.com on

The genetics of maintenance of an all-male lineage in the Squalius alburnoides complex

M. J. A*‡, M. J. C-P*, T. E. D†  M. M. C* *Centro de Biologia Ambiental/Departamento de Zoologia e Antropologia, Faculdade de Cieˆncias, Universidade de Lisboa, C2 – Piso 3, Campo Grande, 1749-016 Lisboa, Portugal and †Department of Biology, Arizona State University, Tempe, Arizona 85287-1501, U.S.A.

(Received 12 July 2001, Accepted 23 January 2002)

Squalius alburnoides is a complex of minnows common to the Iberian Peninsula, consisting of two distinct forms. The most common form is comprised of diploid and polyploid asexual hybrids heterozygous for several diagnostic allozyme loci contributed by Squalius pyrenaicus or Squalius carolitertii and a missing ancestor. The other form is diploid and homozygous for the allele contributed by the missing ancestor at these same loci. Present results from analyses of sex ratio and cytochrome b sequences are not consistent with the evolutionary distinctiveness of this non-hybrid form and suggest that it represents an all-male lineage imbedded within an almost all-female complex. This all-male lineage allowed preservation of the ancestral paternal nuclear genome after the paternal ancestor became extinct in all or most drainages, with important evolutionary implications. 2002 The Fisheries Society of the British Isles

Key words: Squalius alburnoides complex; asexual vertebrates; hybrids; allozymes; cytochrome b.

INTRODUCTION The Iberian minnow Squalius alburnoides (Steindachner) comprises diploid and polyploid forms and is of hybrid origin, incorporating genomes from sympatric bisexual Squalius pyrenaicus (Gu¨nther) in the southern river basins or Squalius carolitertii (Doadrio) in the northern river basins, and that from an undescribed species (Alves et al., 1997a; Carmona et al., 1997). Hybridization in S. alburnoides has altered both oogenesis and spermatogenesis, so that hybrids produce gametes with little or no recombination between the ancestral genomes (Carmona et al., 1997; Alves et al., 1998, 1999). Squalius alburnoides is one of the few examples of asexual (i.e. non-recombinant, Beukeboom & Vrijenhoek, 1998) vertebrates that include both hybrid females and males that are fertile. Hybrid females seem to exhibit distinct reproductive modes according to their geographic origin. Diploid and triploid females from the northern basins reproduce by hybridogenesis, where the carolitertii genome is discarded during oogenesis and replaced in each generation (Carmona et al., 1997). Diploid females from the southern basins transmit clonally their hybrid genome to the eggs, that upon fertilization yield triploid progeny, whereas triploid females ‡Author to whom correspondance should be addressed at present address: Museu Nacional de Histo´ria Natural—Museu Bocage. Rua da Escola Politećnica 58, 1269-102 Lisboa, Portugal. Tel.: +351 21 3921886; fax: +351 21 3969784; email: [email protected] 649 0022–1112/02/030649+14 $35.00/0 2002 The Fisheries Society of the British Isles 650 . .   . present a modified hybridogenesis in which the pyrenaicus genome is discarded in each generation, but the remaining genomes are not transmitted clonally to the eggs as the elimination of the unmatched genome permits ready synapsis of the homospecific genomes (meiotic hybridogenesis, Alves et al., 1998). Diploid hybrid males produce clonal sperm, whereas tetraploid males produce reduced sperm through normal meiosis (Alves et al., 1999). These modes of reproduction imply continuous shifting between forms, where genomes derived from both parental ancestors are cyclically lost, gained or replaced (Alves et al., 2001). In their study of the complex, Carmona et al. (1997) obtained a sample from a single location in the Guadiana River basin that was a mixture of typical hybrid diploid and triploid individuals and a few diploid S. alburnoides-like individuals homozygous at diagnostic allozyme loci for alleles contributed by the missing ancestor (hereafter referred to as ‘ non-hybrid ’ S. alburnoides). Since polymorphic loci of the non-hybrids revealed no significant deviations from Hardy-Weinberg expectations and analysis of mitochondrial (mt) DNA variation indicated a monophyletic relationship between non-hybrids, hybrids, and bisexual S. pyrenaicus, the authors inferred that this non-hybrid form of S. alburnoides was a distinct bisexual species and the maternal ancestor of the hybrid complex. This hypothesis, however, requires further investigation, as genotypic proportions in a small sample like the one used by Carmona et al. (1997) (n=11) are rarely significantly different from those predicted from the Hardy-Weinberg expectations (Lewontin & Cockerham, 1959). In the present study, the origin of the non-hybrid form was further examined by characterizing sex-ratio and mtDNA variation in several collections of non-hybrid and hybrid S. alburnoides and S. pyrenaicus. If the non-hybrid form is an independent, sexually-reproducing lineage as suggested by Carmona et al. (1997), then males and females of this form should be found, and these will form a single, cohesive lineage. Likewise, hybrid S. alburnoides and introgressed S. pyrenaicus should be genetically similar to it, but some specimens of S. pyrenaicus should exhibit phylogenetically distinct mtDNA haplotypes, especially those from drainages where the S. alburnoides complex has not been found.

MATERIAL AND METHODS Specimens of S. alburnoides and S. pyrenaicus were collected from the Sorraia River of the Tejo Basin, the Xe´vora and Degebe Rivers of the Guadiana Basin, and the Odivelas River of the Sado Basin (Fig. 1; Table I). Specimens of S. alburnoides were sexed by dissection and inspection of the gonads, and their liver removed for electrophoresis. Ploidy was determined by flow cytometric analysis of DNA content of erythrocytes, as described in Pro´spero & Collares-Pereira (2000). Each specimen was surveyed at the sAAT* (EC 2.6.1.1) and PGDH* (EC 1.1.1.44) loci. These loci constitute diagnostic markers for the S. alburnoides hybrids, as they show fixed heterozygosity in all populations (Alves et al., 1997a; Carmona et al., 1997). Both non-hybrid and hybrid forms of S. alburnoides were morphologically analysed, and contrasted with the previous results of Collares-Pereira (1984) who described two forms (A and B) that are differentiated by the number of gillrakers and pharyngeal teeth. Form A frequently exhibits a pharyngeal tooth formula of 0,5-5,0 and 12–17 gillrakers (X=15·2), whereas form B tends to have a pharyngeal tooth formula of 0,5-0,4 (rarely 0,5-5,0) and longer and numerous gillrakers (17–26, X=21·3).    .  - 651

Ebro R .

Douro R.

Mondego R. . Tejo R

. 2 1 Guadiana R

3 Sado R. 4 .

Guadalquivir R

F. 1. Map of the Iberian Peninsula showing the distribution of the S. alburnoides complex. The complex is sympatric with S. carolitertii and S. pyrenaicus in the stippled and striped areas, respectively. , Collection sites of the non-hybrid individuals: 1, Sorraia River; 2, Xe´vora River; 3, Degebe River; 4, Odivelas River.

T I. Samples and localities of the populations analysed

No. of Basin River Locality Population specimens

Tejo Sorraia Escusa S. alburnoides 44 S. pyrenaicus 10 Guadiana Xe´vora Ouguela S. alburnoides 40 Degebe Amieira S. alburnoides 33 S. pyrenaicus 10 Sado Odivelas Alvito S. alburnoides 29 S. pyrenaicus 10

Restriction endonuclease analysis (RFLPs) of mt cytochrome (cyt) b gene from up to 10 specimens of each non-hybrid, diploid and polyploid hybrid S. alburnoides and S. pyrenaicus from each drainage was used to quantify patterns of variation within and among drainages and taxa. Methods for PCR ampliﬁcation of cyt b and RFLP analysis are described in Alves et al. (1997b). Five restriction enzymes, BstNI, HaeIII, RsaI, StyI and TaqI, were selected which were found to detect variable sites in S. alburnoides (Alves et al., 1997b). Sequence divergence between haplotypes was estimated from the 652 . .   .

T II. Origin of the cyt b sequences used in the phylogenetic analysis

EMBL/GenBank Species Basin River accession number

S. carolitertii Douro 1. Maça˜s Z75912c 2. Paiva Y10135b 3. Adaja AF045994d Mondego 4. Mondego Z75906c 5. Alva Y10136b S. pyrenaicus Tejo 6. Serta˜ Y10131b 7. Sever Y10132b 8. Sorraia Z75924c 9. Tietar AF045993d Cheleiros 10. Cheleiros AJ427835a Samarra 11. Samarra AJ427836a Guadiana 12. Caia Y10134b 13. Xe´vora Z75927c 15. Estena AF045991d Sado 16. Odivelas Y10133b Z75926c Non-hybrid S. alburnoides Tejo 8. Sorraia AJ427839a Guadiana 13. Xe´vora AJ427838a Sado 16. Odivelas AJ427837a Hybrid S. alburnoides Douro 1. Maça˜s X99431b Mondego 5. Alva X99430b Tejo 6. Serta˜ X99429b 7. Sever X99427b 8. Sorraia X99428b Guadiana 12. Caia X99426b 13. Xe´vora X99421b 14. Degebe X99425b 15. Estena AF045992d Sado 16. Odivelas X99432b C. arcasii Douro 1. Maça˜s X99424b C. lemmingii Guadiana 12. Caia X99423b A. hispanica Guadiana 13. Xe´vora X99422b

aPresent study; bAlves et al. (1997b); cBrito et al. (1997); dZardoya & Doadrio (1998). proportion of shared sites using the maximum-likelihood algorithm of Nei & Tajima (1983), using the software package REAP 4.0 (McElroy et al., 1992). Partitioning of mtDNA variation was accomplished by a hierarchical analysis of molecular variance, AMOVA (Excoffier et al., 1992), using the software package ARLEQUIN 2.000 (Schneider et al., 2000). For one nuclear non-hybrid S. alburnoides specimen randomly chosen from each drainage, the entire cyt b gene was amplified and sequenced as described in Alves et al. (1997b). Sequences were aligned manually using the MacDNASIS 2.0 alignment editor to one sequence of hybrid S. alburnoides (EMBL accession number X99421). Maximum parsimony (MP) analysis was conducted using PAUP* (Swofford, 1998). In addition to the nuclear non-hybrid specimens, the ingroup included all cyt b sequences of hybrid S. alburnoides, S. pyrenaicus and S. carolitertii available in the EMBL and GenBank databases, and also one sequence from each of the sympatric related cyprinids Chondrostoma arcasii (Steindachner), Chondrostoma lemmingii (Steindachner), and Anaecypris hispanica (Steindachner) (Table II). Two specimens of S. pyrenaicus from the    .  - 653

T III. Number of diploid S. alburnoides per allozyme genotypes at the diagnostic loci

Sorraia River Xe´vora River Degebe River Odivelas River NH 7 H 7 H 8 NH 7 H 8 NH 7 H 8 NH 7 H 8

Genotypes sAAT* a/a 7 0 0 7 0 19 0 9 0 p/a 0 10 5 0 3 0 6 0 5 PGDH* a/a 7 0 0 7 0 18 0 9 0 p1/a 0 8 5 0 3 1 0 0 5 p2/a 0 2 0 0 0 0 6 0 0

NH, Non-hybrid form; H, hybrid form. ‘ p ’ and ‘ a ’ are the alleles attributed to S. pyrenaicus and to the other ancestor, respectively.

Samarra and Cheleiros drainages (where the S. alburnoides complex does not occur) were also sequenced and included in the phylogenetic analysis. The North American cyprinid Notemigonus crysoleucas (Mitchill) (GenBank accession number U01318) was used as the outgroup. Topologies were generated by heuristic search, using the tree bisection- reconnection method with 50 random addition sequence replicates and characters weighted equally. Support for speciﬁc nodes was examined by generation of 1000 bootstrap replicates using the parameters above, except that simple addition of taxa was used, with one tree held per step. Most parsimonious trees were compared with those expected from alternate hypotheses by comparing speciﬁc topologies as described by Templeton (1983) and implemented by PAUP*.

RESULTS SAMPLE CHARACTERIZATION Flow cytometry indicated that diploids were common in most samples of S. alburnoides, representing 50, 27, 86 and 48% of the individuals collected from the Sorraia, Xe´vora, Degebe and Odivelas Rivers, respectively (Table III). They included both females and males, with males predominating in all diploid sub-samples. Polyploids included mainly triploid females, but also some rare tetraploid females and males in the Sorraia sample. All diploid females and polyploid females and males displayed heterozygous patterns at the diagnostic loci, sAAT* and PGDH*, suggesting that they are hybrids. In contrast, all diploid males collected from the Xe´vora, Degebe and Odivelas Rivers were homozygous at both diagnostic loci with the exception of a single male from the Degebe River, which was heterozygous for the S. pyrenaicus allele at PGDH* (Table III). In the Sorraia sample, 10 diploid males showed hybrid patterns, whereas the remaining seven diploid males were homozygous. None of the 42 specimens homozygous at both diagnostic loci were female. Non-hybrid S. alburnoides possessed 17–23 gillrakers and most commonly exhibited pharyngeal tooth counts of 0,5-4,0, consistent with the values attributed to form B by Collares-Pereira (1984). Hybrid diploid and polyploid 654 . .   .

S. alburnoides showed 13–17 gillrakers and a variety of pharyngeal tooth formulae, 0,5-5,0, 0,5-5,1, 1,5-5,0, 1,5-5,1 and 1,5-5,2, most similar to form A of Collares-Pereira (1984).

DISTRIBUTION OF mtDNA VARIATION RFLPs analysis of mt cyt b from up to 10 specimens of each diploid non-hybrid and diploid and polyploid hybrid S. alburnoides and S. pyrenaicus from each basin yielded nine composite haplotypes (Table IV). Non-hybrids shared cyt b haplotypes with both hybrid S. alburnoides and S. pyrenaicus from the same basin. The exception was a single male from the Sado Basin which showed a unique haplotype, differing by two site changes from other nuclear non-hybrids and one site change from hybrid S. alburnoides and S. pyrenaicus in the same sample. Analysis of molecular variance (AMOVA) revealed that variation within river basins was low, accounting for only 10·6% of total variance. When molecular variance was partitioned into two hierarchical levels (river basins and forms), variation among basins was high and significant (CT=0·896, P<0·001) while variation among forms within basins was low and not significant (SC= 0·064, P=0·8422).

PHYLOGENETIC RELATIONSHIPS Parsimony analysis of cyt b gene sequences using phylogenetically informative characters yielded 205 equally parsimonious trees, requiring a minimum of 450 mutational steps (consistency index=0·600, retention index=0·742). The top- ology indicates a monophyletic relationship among non-hybrid S. alburnoides, hybrid S. alburnoides and all S. pyrenaicus relative to other genera examined (Fig. 2). Bootstrap support for this clade is high, occurring in 96% of the replicates. Within this clade, there is strong support for monophyly of samples from the same drainage, independent of their nuclear genomic composition. Hybrid S. alburnoides from the northern basins (Douro and Mondego) are closely related to S. alburnoides and S. pyrenaicus from the Tejo and Guadiana Basins. Allopatric samples of S. pyrenaicus from Samarra and Cheleiros Basins are included in a monophyletic lineage comprised mostly of samples from the Tejo Basin, however, this grouping was only recovered in 54% of the bootstrap replicates. These allopatric samples are imbedded well within the S. alburnoides+S. pyrenaicus complex as part of well-supported monophyletic lineage that includes Guadiana, Tejo, and northern drainages (87% of bootstrap replicates). Alternative hypotheses for the origin of non-hybrid S. alburnoides were examined by constraining trees to topologies predicted from hypothesized modes of origin, and contrasting these trees with the most parsimonious trees described above. In the first set of constrained trees all S. alburnoides, both hybrid and non-hybrid, were constrained to form a monophyletic group, with all S. pyrenaicus as the sister group. This would be consistent with the hypothesis that the non-hybrid form is the maternal ancestor of the hybrid complex, S. pyrenaicus the paternal ancestor, without introgression between forms. Heuristic search under this constraint identified 96 trees of 477 steps. In the second set of constrained trees, nuclear non-hybrid S. alburnoides were forced to be T IV. Matrix of presence or absence of eight polymorphic restriction sites defining nine cyt b haplotypes found in specimens of non-hybrid (NH) and hybrid (HYB) S. alburnoides, and S. pyrenaicus (PYR). Nucleotide positions (5 end) of each restriction site for each enzyme are in order: BstNI—363, 894; HaeIII—378; RsaI—394, 666, 898; TaqI—178, 928. Haplotype designation followed Alves et al. (1997b)

Restriction site matrix Number of each haplotype per sample Sorraia River Degebe River Odivelas River Haplotype BstNI HaeIII RsaI TaqI (Tejo Basin) (Guadiana Basin) (Sado Basin) NH HYB* PYR NH HYB* PYR NH HYB* PYR

VII 01 0 010 01 7 10 9 VIII 1 1 0 0 1 0 0 1 7 6 6 IX 11 0 000 01 2 1 X 11 0 011 01 3 1 2 XI 10 0 011 11 1 1 XII 11 1 100 00 6 9 8 XIII 1 0 1 1 0 0 0 0 1 2 XIV 01 1 010 01 1 XV 10 1 100 01 1

*Data already presented in Alves et al. (1997b). 656 . .   .

HYB1 Northern 86 HYB5 78 HYB6 PYR7 HYB7 HYB8 Tejo 54 NH8 87 PYR6 PYR8 PYR9 PYR10 - Cheleiros PYR11 - Samarra S. alburnoides + HYB13 S. pyrenaicus 85 NH13 PYR12 96 79 HYB12 Guadiana HYB14 54 HYB15 56 PYR13 PYR15 HYB16 100 91 NH16 99 Sado PYR16 PYR16 CAR1 98 70 CAR2 100 CAR3 S. carolitertii 82 CAR5 CAR4 74 C. lemmingii C. arcasii A. hispanica Notemigonus F. 2. Strict consensus of 205 most parsimonious trees (450 steps, consistency index=0·600, retention index=0·742) resolved from a heuristic analysis (see text) of complete 1140 bp cytochrome b sequences. Numbers on the nodes represent the per cent of 1000 bootstrap replicates in which the particular node was monophyletic in the strict consensus tree. In the designations of the specimens, NH, HYB, CAR and PYR refer to non-hybrid and hybrid S. alburnoides, S. carolitertii and S. pyrenaicus, respectively, and numbers refer to collection river (see Table II). monophyletic exclusive of all other taxa. This would be consistent with the hypothesis that the non-hybrid form represents a bisexual species that is the paternal ancestor of the hybrid form, S. pyrenaicus is the maternal ancestor, without introgression between forms. Heuristic search under this constraint identified 60 trees of 486 steps. Statistical contrasts revealed that most parsimonious trees were significantly better (P<0·0001) than both sets of constrained trees. In another alternative hypothesis, allopatric samples of S. pyrenaicus were forced to be the sister group of all the other samples of the S. alburnoides+S. pyrenaicus complex. This pattern is consistent with the prediction that the    .  - 657 mtDNA in the paternal S. pyrenaicus was completely replaced by introgressive hybridization from the maternal non-hybrid S. alburnoides wherever the two co-occur. Heuristic search under this constraint recovered 256 trees, requiring a minimum of 456 steps. While this alternative hypothesis requires only six additional steps, significance values from tests that the most parsimonious trees were shorter than the 256 constrained trees were always low (P<0·10) and sometimes significant (P<0·05).

DISCUSSION Results from analysis of sex ratio of the four samples of non-hybrid S. alburnoides representing three basins are not consistent with the evolutionary distinctiveness of this form. All non-hybrid specimens collected were male, suggesting that they are not an independent, sexually reproducing lineage. Low levels of mtDNA variation within drainages and strength of support for monophyly of local drainage groups of non-hybrid males, hybrids and S. pyrenaicus seem to corroborate this result. The most likely explanation for geographical consistency of mtDNA variation across all three forms is that they share recent common ancestry, most likely through multiple, independent origins of S. alburnoides from female S. pyrenaicus and an unidentified male progenitor (Alves et al., 1997b). Under this model, the non-hybrid form is most likely a product of the asexual hybrids, exhibiting pyrenaicus-like mtDNA while possess- ing the nuclear genome of the paternal ancestor. Statistical contrasts of topologies predicted from the alternate modes of origin of the non-hybrids also suggest that they are not an independent bisexual form acting as the maternal or paternal contributor to this complex. Although the hypothesis of the non-hybrid form as the maternal ancestor with extensive introgression between it and S. pyrenaicus wherever the two co-occur (third set of constrained trees) was statistically not falsified, it seems unlikely due to the high bootstrap values placing allopatric S. pyrenaicus well within the S. alburnoides+S. pyrenaicus complex. Examination of other populations is also inconsistent with this prediction, especially since introgression is limited in the Douro and Mondego river basins (Brito et al., 1997), where S. carolitertii provides sperm for the complex. The two genetically distinct forms of S. alburnoides are readily identifiable through morphological characters, corresponding to the two forms defined by Collares-Pereira (1984): form A includes individuals with hybrid genomes independently of their ploidy level, whereas form B comprises diploid nuclear non-hybrid specimens. The sex-ratio of the non-hybrid form observed in the present study (all-male) is consistent with the data of Collares-Pereira (1984) who found no females among 192 specimens of form B captured in the Tejo, Guadiana, and Sado river basins. A single male from the Degebe River exhibited morphological values that classify it as non-hybrid but was heterozygous for the S. pyrenaicus allele at one diagnostic loci, the PGDH*. Such variation may have resulted from recombination between the parental genomes in the hybrid ancestor. Although recombinational events between parental genomes are very rare in unisexual lineages, evidence for limited recombination has been provided by studies in 658 . .   .

Ambystoma jeffersonianum-laterale (Bogart, 1989), Rana esculenta (Uzzell et al., 1977; Graf & Polls Pelaz, 1989; Plo¨tner & Klinkhardt, 1992), Cnemidophorus tesselatus (Parker et al., 1989), and Cnemidophorus lemniscatus (Sites et al., 1990). Shared alleles between S. pyrenaicus and the non-hybrid males may, however, be a consequence of common inheritance, mutation or intragenic recombination. Experimental matings (Alves et al., 1998) provide clues as to how nuclear non-hybrid males might be produced in present-day populations. Triploid females of S. alburnoides from the Tejo and Guadiana Basins frequently produce haploid eggs by meiotic hybridogenesis, excluding the S. pyrenaicus (P) genome and transmitting genetically diverse gametes that result from recombination of the two homospecific genomes from the unknown ancestor (A genome). When these eggs are fertilized by a haploid sperm produced meiotically by AA males (=non-hybrid S. alburnoides), only AA males are produced. When these eggs are fertilized by a P sperm produced meiotically by S. pyrenaicus males, hybrid PA females and males are produced. The reason for the absence of AA females is difficult to understand as the mechanism of sex determination in the S. alburnoides complex is not yet well understood. Although cytological data indicated the maternal species S. pyrenaicus has a ZW female/ZZ male sex chromosome heteromorphism (Collares-Pereira et al., 1998), there is evidence that non-W-linked genes are involved in sex determination, and these genes are expressed differently in hybrid and non-hybrid genome combinations (Alves et al., 1998). Based on current information (Alves et al., 1997a, b, 1998, 2001 and present paper), it is possible to construct a hypothetical model that outlines the evolutionary trajectory of the all-male form within the hybrid complex (Fig. 3). The complex had independent origins in each of the southern drainages (Tejo, Guadiana and Sado) through matings between an undescribed paternal ancestor and S. pyrenaicus. Early in the progression, triploid PAA females would have been produced from clonal PA females through matings with the paternal ancestor (AA males with A mtDNA). When these triploid hybrid females mated with males from paternal ancestor they would have produced only diploid non-hybrid males (AA with P mtDNA). This would have initiated a cycle that maintains this nuclear non-hybrid male lineage, where the diploid and triploid hybrid females act as hosts of the A genome. New P genomes have to be recruited from the S. pyrenaicus population continuously to maintain diploid and triploid hybrid females in the cycle. This cycle might have been effectively isolated from the paternal ancestor at the outset; alternatively, the cycle could have become isolated with loss of the paternal progenitor. Production of specimens with a monospecific nuclear genome from hybrid parents is known to occur in other hybrid complexes, namely in R. esculenta (Spolsky & Uzzell, 1984; Hotz et al., 1992) and in Phoxinus eos-neogaeus (Cope) (Goddard & Schultz, 1993). In these complexes, the ‘ reconstituted ’ specimens may act, through backcrosses with the parental species, as a bridge for interspecies transfer of mtDNA and eventually of nuclear genes (if recombination between the parental genomes have occurred in the hybrid lineage) (Dowling & Secor, 1997) or regenerate sexually reproducing populations (Vrijenhoek, 1998). However, nuclear non-hybrid males of the S. alburnoides    .  - 659

S. pyrenaicus Unknown ancestor

PPP × AAA Original hybridization event

P A

Diploid hybrid PAP × AAA Non-reductional division PA A

Triploid hybrid PAAP × AAA Meiotic hybridogenesis AA A A PPP × PAAP

PAAP × AAP P A AA

AA A A PAP PAAP and and AAP

PAP × AAP Non-hybrid male lineage

PA A

PAAP

F. 3. Hypothetical evolutionary trajectory of the all-male non-hybrid lineage within the hybrid complex: above dash line, mechanisms of the origin of the hybrid complex; below dash line, perpuating mechanisms of the complex. P and A represent the genome of S. pyrenaicus andofthe other ancestor, respectively. The main letters indicate the nuclear genomes, whereas the prime letters indicate the mitochondrial genome. complex seem to be unique, as they essentially represent a stable all-male lineage nested within an almost all-female lineage. Because meiosis occurs in this male lineage (Alves et al., 1998), it represents a potential source of genetic variation in 660 . .   . the hybrid complex. Nevertheless, the genetic variability of nuclear non-hybrid male S. alburnoides should be lower than that of a bisexual population, since the A lineage must pass through a non-recombining unisexual host stage (PA) that should reduce its variability relative to a typical bisexual population. It could be expected that the all-male lineage might eventually allow recurrent synthesis of new hybrid asexual lineages, if non-hybrid males mated with S. pyrenaicus females. The viability of such crosses, however, needs to be experimentally tested, as the capacity of founding asexual lineages seems to be a property of only some populations of the ancestral species (Hotz et al., 1985; Densmore et al., 1989; Quattro et al., 1992). Although sexually reproducing populations of the paternal ancestor may still exist, namely in some unexplored independent small river basins, the present study suggests that in the three basins surveyed the paternal species is most likely extinct. In these basins, the all-male lineage allowed preservation of the ancestral paternal nuclear genome. The non-hybrid males are apparently a significant piece in the dynamics of these hybrid populations, ‘ playing the role ’ of the probably extinct paternal ancestor. We are grateful to A. Pires, who firstly called our attention the S. alburnoides-like males from the Xe´vora River. Special thanks are also due to I. Pro´spero and L. da Costa for ploidy determination and assistance in sampling. We thank J. Bogart for his helpful comments on an early draft of the manuscript. We acknowledge Direcçaõ Geral das Florestas for permission to collect specimens. This work was supported by Fundaçaõ para a Cieˆncia e Tecnologia (PRAXIS/P/BIA/11086/1998). MJA received the Ph.D. grant PRAXIS XXI/BD/5735/95.

References

Alves, M. J., Coelho, M. M. & Collares-Pereira, M. J. (1997a). The Rutilus alburnoides complex (Cyprinidae): evidence for a hybrid origin. Journal of Zoological System- atics and Evolutionary Research 35, 1–10. Alves, M. J., Coelho, M. M., Collares-Pereira, M. J. & Dowling, T. E. (1997b). Maternal ancestry of the Rutilus alburnoides complex (Teleostei, Cyprinidae) as determined by analysis of cytochrome b sequences. Evolution 51, 1584–1592. Alves, M. J., Coelho, M. M. & Collares-Pereira, M. J. (1998). Diversity in the reproductive modes of females of the Rutilus alburnoides complex (Teleostei, Cyprinidae): a way to avoid the genetic constraints of uniparentalism. Molecular Biology and Evolution 15, 1233–1242. Alves, M. J., Coelho, M. M., Pro´spero, M. I. & Collares-Pereira, M. J. (1999). Production of fertile unreduced sperm by hybrid males of the Rutilus alburnoides complex (Teleostei, Cyprinidae): an alternative route to genome tetraploidization in unisexuals. Genetics 151, 277–283. Alves, M. J., Coelho, M. M. & Collares-Pereira, M. J. (2001). Evolution in action through hybridisation and polyploidy in an Iberian freshwater ﬁsh: a genetic review. Genetica 111, 375–385. Beukeboom, L. W. & Vrijenhoek, R. C. (1998). Evolutionary genetics and ecology of sperm-dependent parthenogenesis. Journal of Evolutionary Biology 11, 755–782. Bogart, J. P. (1989). A mechanism for interspeciﬁc gene exchange via all-female salamander hybrids. In Evolution and Ecology of Unisexual Vertebrates (Dawley, R. M. & Bogart, J. P., eds), pp. 170–179. Albany: New York State Museum. Brito, R. M., Briolay, J., Galtier, N., Bouvet, Y. & Coelho, M. M. (1997). Phylogenetic relationships within genus Leuciscus (Pisces, Cyprinidae) in Portuguese fresh waters, based on mitochondrial DNA cytochrome b sequences. Molecular Phylogenetics and Evolution 8, 435–442.    .  - 661

Carmona, J. A., Sanjur, O. I., Doadrio, I., Machordom, A. & Vrijenhoek, R. C. (1997). Hybridogenetic reproduction and maternal ancestry of polyploid Iberian fish: the Tropidophoxinellus alburnoides complex. Genetics 146, 983–993. Collares-Pereira, M. J. (1984). The ‘ Rutilus alburnoides (Steindachner, 1866) complex ’ (Pisces, Cyprinidae). I. Biometrical analysis of some Portuguese populations. Arquivos do Museu Bocage (Se´rie A) 2, 111–143. Collares-Pereira, M. J., Pro´spero, M. I., Bileú, R. I. & Rodrigues, E. M. (1998). Leuciscus (Pisces, Cyprinidae) karyotypes: transect of Portuguese populations. Genetics and Molecular Biology 21, 63–69. Densmore, L. D. III, Moritz, C. C., Wright, J. W. & Brown, W. M. (1989). Mitochondrial-DNA analyses and the origin and relative age of parthenogenetic lizards (genus Cnemidophorus). IV. Nine sexlineatus-group unisexuals. Evolution 43, 943–983. Dowling, T. E. & Secor, C. L. (1997). The role of hybridization and introgression in the diversification of animals. Annual Reviews of Ecology and Systematics 28, 593–619. Excoffier, L., Smouse, P. E. & Quattro, J. M. (1992). Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479–491. Goddard, K. A. & Schultz, R. J. (1993). Aclonal reproduction by polyploid members of the clonal hybrid species Phoxinus eos-neogaeus (Cyprinidae). Copeia 1993, 650–660. Graf, J.-D. & Polls Pelaz, M. (1989). Evolutionary genetics in the Rana esculenta complex. In Evolution and Ecology of Unisexual Vertebrates (Dawley, R. M. & Bogart, J. P., eds), pp. 289–301. Albany: New York State Museum. Hotz, H., Mancino, G., Bucci-Innocenti, S., Ragghianti, M., Berger, L. & Uzzell, T. (1985). Rana ridibunda varies geographically in inducing clonal gametogenesis in interspecies hybrids. Journal of Experimental Zoology 236, 199–210. Hotz, H., Beerli, P. & Spolsky, C. (1992). Mitochondrial DNA reveals formation of nonhybrid frogs by natural matings between hemiclonal hybrids. Molecular Biology and Evolution 9, 610–620. Lewontin, R. C. & Cockerham, C. C. (1959). The goodness-of-fit test for detecting natural selection in random mating populations. Evolution 53, 561–564. McElroy, D., Moran, P., Bermingham, E. & Kornfield, I. (1992). REAP: an integrated environment for the manipulation and phylogenetic analysis of restriction data. Journal of Heredity 83, 157–158. Nei, M. & Tajima, F. (1983). Maximum likelihood estimation of the number of nucleotide substitutions from restriction sites data. Genetics 105, 207–217. Parker, E. D. Jr, Walker, J. M. & Paulissen, M. A. (1989). Clonal diversity in Cnemidophorus: ecological and morphological consequences. In Evolution and Ecology of Unisexual Vertebrates (Dawley, R. M. & Bogart, J. P., eds), pp. 72–86. Albany: New York State Museum. Plo¨tner, J. & Klinkhardt, M. (1992). Investigations on the genetic structure and the morphometry of a pure hybrid population of Rana kl. esculenta (Anura, Ranidae) in North Germany. Zoologischer Anzeiger 229, 163–184. Pro´spero, M. I. & Collares-Pereira, M. J. (2000). Nuclear DNA content variation in the diploid-polyploid Leuciscus alburnoides complex (Teleostei, Cyprinidae) assessed by flow cytometry. Folia Zoologica 49, 53–58. Quattro, J. M., Avise, J. C. & Vrijenhoek, R. C. (1992). Mode of origin and sources of genotypic diversity in triploid fish clones (Poeciliopsis: Poeciliidae). Genetics 130, 621–628. Schneider, S., Roessli, D. & Excoffier, L. (2000). Arlequin ver. 2.000: A Software for Population Genetics Data Analysis. Geneva: Genetics and Biometry Laboratory, University of Geneva. Sites, J. W. Jr, Peccinini-Seale, D., Moritz, C., Wright, J. W. & Brown, W. M. (1990). The evolutionary history of the parthenogenetic Cnemidophorus lemniscatus (Sauria, Teiidae). I. Evidence for a hybrid origin. Evolution 44, 906–921. 662 . .   .

Spolsky, C. & Uzzell, T. (1984). Natural interspecies transfer of mitochondrial DNA in amphibians. Proceedings of the National Academy of Sciences, USA 81, 5802– 5805. Swoﬀord, D. L. (1998). PAUP*: Phylogenetic Analysis Using Parsimony and Other Methods. Version 4. Sunderland, MA: Sinauer Associates. Templeton, A. R. (1983). Phylogenetic inference from restriction cleavage site maps with particular reference to the evolution of humans and the apes. Evolution 37, 221–244. Uzzell, T. M., Gu¨nther, R. & Berger, L. (1977). Rana ridibunda and Rana esculenta,a leaky hybridogenetic system (Amphibia: Salientia). Proceedings of the Academy of Natural Sciences of Philadelphia 128, 147–171. Vrijenhoek, R. C. (1998). Clonal organisms and the beneﬁts of sex. In Advances in Molecular Ecology (Carvalho, G. R., ed.), pp. 151–172. Amsterdam: IOS Press. Zardoya, R. & Doadrio, I. (1998). Phylogenetic relationships of Iberian cyprinids: systematic and biogeographical implications. Proceedings of the Royal Society of London B 265, 1365–1372.