phylogenetic evidence for role-reversals of gender-associated

Phylogenetic Evidence for Role-Reversals of Gender-Associated Mitochondrial DNA in Mytilus (Bivalvia: Mytilidae)

Walter R. Hoeh, *cl Donald T. Stewart,* Carlos Saavedra,*2 Brent W. Sutherland,*3 and Eleftherios Zouros* j- *Department of Biology, Dalhousie University; and TDepartment of Biology, University of Crete and Institute of Marine Biology of Crete, Greece

Distinct gender-associated mitochondrial DNA (mtDNA) lineages (i.e., lineages which are transmitted either through males or through females) have been demonstrated in two families of bivalves, the Mytilidae (marine mussels) and the Unionidae (freshwater mussels), which have been separated for more than 400 Myr. The mode of transmission of these M (for male-transmitted) and F (for female-transmitted) molecules has been referred to as doubly uniparental inheritance (DUI), in contrast to standard maternal inheritance (SMI), which is the norm in animals. A previous study suggested that at least three distinct origins of DUI are required to explain the phylogenetic pattern of M and F lineages in freshwater and marine mussels. Here we present phylogenetic evidence based on partial sequences of the cytochrome c oxidase subunit I gene and the 16s RNA gene that indicates that DUI is a dynamic phenomenon. Specifically, we demonstrate that F lineages in three species of Mytilus mussels, M. edulis, 44. trossulus, and M. californianus, have spawned separate lineages which are now associated only with males. This process is referred to as “masculinization” of F mtDNA. By extension, we propose that DUI may be a primitive bivalve character and that periodic masculinization events combined with extinction of previously existing M types effectively reset the time of divergence between conspecific gender-associated mtDNA lineages.

Introduction

The phenomenon of doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA) has now been documented in several phylogenetically diverse bivalve taxa. These taxa include marine mussels of the family Mytilidae (Myti2u.s edu2is [Skibinski, Gallagher, and Beynon 1994a, 1994b; Zouros et al. 1994a, 1994b], M. trossulus [Geller 1994; Zouros et al. 1994b; Rawson and Hilbish 1995; Stewart et al. 19951, M. galloprovincialis [Rawson and Hilbish 1995; Quesada, Skibinski, and Skibinski 1996; Saavedra, Reynero, and Zouros 19971, Geukensia demissa [Hoeh et al. 19961) and freshwater mussels of the family Unionidae (Pyganodon fragilis, P. grandis and Fusconaia Jlava [Liu, Mitton, and Wu 1996; Hoeh et al. 19961). In these bivalves, males possess two distinct classes of mtDNA: an M type, so-named because it is transmitted from male parents to their sons, and an F type that is transmitted from generation to generation through females. Although sons also receive their mother’s F type mtDNA, they do not transmit it to their offspring.

Rawson and Hilbish (1995) and Stewart et al. ( 1995) were the first to examine DUI from a phylogenetic perspective. Using mitochondrial 16s RNA gene sequences from M. edulis, M. trossulus, and A4. galloprovincialis, Rawson and Hilbish (1995) showed that

I Present address: Department of Zoology, Miami University.

2 Present address: Department of Biology, University of Crete.

3 Present address: Department of Microbiology and Immunology, University of British Columbia.

Key words: phylogenetics, mitochondrial DNA, doubly uniparental inheritance, cytochrome c oxidase I, 16s RNA, masculinization, Mytilus, Bivalvia, Mytilidae.

Address for correspondence and reprints: Donald T. Stewart, De- partment of Biology, Fairfield University, Fairfield, CT 06430-5 195. E-mail: [email protected].

Mol. Bid. Evol. 14(9):959-967. 1997 0 1997 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

the F sequences and the M sequences from all three species formed two gender-associated groups. The same result was observed by Stewart et al. (1995) in a collection of cytochrome c oxidase subunit III (COIII) sequences from M. edulis and M. trossulus. Both studies concluded that the formation of distinct F and M lineages must have preceded the emergence of the three species of mussels from their common ancestor.

The discovery of DUI in freshwater mussels (Liu, Mitton, and Wu 1996) prompted Hoeh et al. (1996) to examine the phylogeny of M and F mitotypes in both marine and freshwater mussels. Specifically, these au- thors compared CO1 sequences from six taxa: M. edulis, M. trossulus, G. demissa, P. grandis, P. fragilis, and F. Java. As expected from the previous analysis of 16s RNA (Rawson and Hilbish 1995) and CO111 sequences (Stewart et al. 1995), Hoeh et al. (1996) obtained distinct M and F groupings for the Myths mitotypes (fig. 1). The six M and F sequences of the three unionid species (P. grandis, P. fragilis, and F. Java) also formed two distinct groups defined on the basis of gender, but as a whole, the freshwater mussel sequences formed a group that was distinct from the marine mussel sequences. The M and F types present in Geukensia formed a sister group to the other mytilid sequences. The com- plete phylogenetic tree of the 12 sequences indicated at least three separate divergences of M and F mitotype lineages, one in each of the lines leading to Myths and to Geukensia and one in the line leading to freshwater mussels (fig. 1). Hoeh et al. (1996) inferred from these three F/M mitotype divergence events either that there have been multiple origins of DUI in bivalves or that there was a single origin of DUI in the ancestral bivalve lineage but reversals in the route of mitotype transmission had mimicked de novo divergences of F and M lineages. Specifically, Hoeh et al. (1996) referred to the hypothetical spawning of an M type from an F type as a “masculinization” event. The corresponding switch

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r F. flava M

3 P. grandis M

+ L P. fragilis M

-k

F. flava F

P. grandis F

P. fragilis F

r G. demissa M

1 /L. damissa F

-2 M. edulis M

M. trossulus M f

M. edulis F

M. trossulus F

FIG. l.-Topology from Hoeh et al. (1996) indicating three divergence events between M and F mitotypes (shown by arrows) in a collection of freshwater and marine mussels. The tree suggests that one divergence event occurred in an ancestor to the freshwater mussel species P. grandis, P. fragilis, and F. Jlava, another in the lineage leading to the marine mussel G. demissa, and one in a common ancestor to M. edulis and M. trossulus.

from an M to an F type would be referred to as a “feminization” event. This hypothesis was based on obser- vations that the fidelity of DUI is not always perfect. That is, females containing an M type and males lacking an M type have occasionally been observed (Fisher and Skibinski 1990; Zouros et al. 1992, 1994b; Saavedra, Reynero, and Zouros 1997). Such anomalous individuals could provide information about how an mtDNA type associated with otie gender lineage could switch its route of transmission to the opposite gender lineage.

To further evaluate the alternate hypotheses indicated above, we have characterized additional mitotype lineages within Mytilus for CO1 and 16s RNA and phylogenetically analyzed these sequences in conjunction with the CO1 sequences presented in Hoeh et al. (1996) and the 16s RNA sequences presented in Rawson and Hilbish (1995). From these analyses, we present evidence that M mitotypes in the genus Mytilus are derived from multiple ancestral lineages. We then demonstrate how the reversal of the transmission route of F genomes (i.e., masculinization events) best accounts for the observed phylogenetic pattern, and we suggest a sequence of events to explain the groupings of M and F types at different levels of the bivalve mitochondrial phylogeny.

Materials and Methods Sequencing Protocol

Total DNA was isolated from either gonad or spawned gametes of both sexes of Mytilus californianus. This material was used to amplify a 710-bp fragment of the mitochondrial cytochrome oxidase subunit I gene (COI) using the primers LCO1490 and HC02198 (for primer sequences and details of the amplification protocol see Folmer et al. [1994] and Hoeh et al. [ 19961). These primers were also used to generate 622 bp of CO1 sequence either by cycle sequencing (AmpliCycle Se-

quencing Kit, Perkin Elmer) or by direct sequencing (Sequenase version 2.0 DNA Sequencing Kit and Re- agent Pack, USB) of the PCR product. Approximately 590 bp of sequence was obtained using either primer. Consequently, 85%-90% of the total sequence obtained was confirmed by sequencing in both directions.

We have also obtained additional CO1 sequences of two M. trossulus mitotypes and one M. edulis mitotype (designated M-O, F-O, and M-A, respectively). M-O and M-A were classified as M types because they were isolated from the gonads of males (Saavedra, Reynero, and Zouros 1996). Restriction mapping (data not shown) indicated that these putative mitochondrial DNA types were of a size consistent with mitochondrial genomes and therefore were not the result of transfer of mitochondrial genes to the nucleus. Although the restriction fragment profiles also indicated that M-A and M-O were considerably divergent from the most common M types of M. edulis and M. trossulus, respectively, these mitotypes were not previously sequenced because of tech- nical difficulties (see Stewart et al. 1995). Briefly, because males are heteroplasmic for an M and an F type and because of the sequence similarity of M-O and M-A with their conspecific F types (see below), it was difficult to isolate the CO111 amplification products of M-O and M-A from the amplification products of their ac- companying F types. The newly obtained CO1 sequences were manually aligned, using MacClade (Maddison and Maddison 1992), with CO1 sequences previously analyzed by Hoeh et al. (1996).

We also amplified and obtained sequence from a 527-bp portion of the mitochondrial 16s RNA gene from two male and two female M. californianus specimens using the primer pair 16s AR and 16s BR (Pal- umbi et al. 199 1; Rawson and Hilbish 1995). For an outgroup, we sequenced the F type from two female Geukensia demissa. The amplification protocol followed Rawson and Hilbish (1995) and the product was sequenced directly as described above. These sequences were aligned against 458 bp of 16s RNA sequence of A4. edulis and M. trossulus M and F types extracted from the GenBank database (U22864, U22865, U22879, and U22882; Rawson and Hilbish 1995).

The CO1 and 16s RNA sequences generated herein have been submitted to the GenBank database (acces- sion numbers U68770-U68772 [16S RNA] and U68773-U68777 [COI]).

Phylogenetic Reconstruction

In this paper, we present three sets of phylogenetic reconstructions. The first set was conducted to test the validity of the deeper nodes of our previously reported topology (see Hoeh et al. 1996, fig. 1). To this end, we conducted maximum-parsimony (MP) (PAUP version 3.1.1; Swofford 1993) and neighbor-joining (NJ) (MEGA version 1.02; Kumar, Tamura, and Nei 1993) analyses on the inferred amino acid sequences of our CO1 data set (i.e., 11 mytilid and 6 unionid sequences). The MP analysis was conducted with and without invoking a step matrix to weight amino acid substitutions relative to the minimum number of changes required to

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switch from one amino acid to another. NJ trees were constructed using an uncorrected amino acid distance matrix and matrices for which distances were estimated using gamma parameters (a) of 0.5, 1.0, and 2.0, respectively.

In the second set of analyses, we focus on the relationship among the nine M and F Mytilus sequences using the CO1 nucleotide data. The methods used in- cluded MP, NJ and maximum likelihood (ML) (DNAML, PHYLIP version 3.5~; Felsenstein 1993). The ML analysis was performed using 100 random terminal mitotype addition sequence runs with the global rear- rangement option in force. The third set of analyses was conducted on a subset of the mytilid sequences for which a combined CO1 and 16s RNA nucleotide data set was available. Although the CO1 and 16s RNA sequences for a given mitotype (e.g., M. trossulus M) were not obtained from the same individual, it is assumed that the two sequences that were pooled to form a composite mitotype share a more recent common ancestor with each other than they do with any other sequence includ- ed in the analysis. Again, all three methods of tree reconstruction were used. Percent bootstrap support for the resulting nodes was evaluated using 1,000 replica- tions for all of the MP and NJ analyses and 100 repli- cations for the ML analysis (Hillis and Bull 1993).

In the second set of analyses, we used methods that take into consideration different rates of sequence divergence among sites and different transition (TS) and transversion (TV) ratios across codon positions within the CO1 gene. The ML and NJ analyses also corrected for differences in frequencies of the four nucleotides. Specifically, the NJ tree was reconstructed from a distance matrix estimated using the Tamura-Nei model (Ta- mura and Nei 1993) and a gamma coefficient of 1 .O. In the third set of analyses, we were also able to compensate for the difference in the overall level of sequence divergence between the CO1 and 16s RNA genes in the MP and ML analyses. These weighting schemes were implemented to give more weight to presumably more conservative changes (Hillis, Huelsenbeck, and Cun- ningham 1994) and/or to estimate, as accurately as possible, the evolutionary distance between mitotypes (Ku- mar, Tamura, and Nei 1993).

The approximate number of changes at each CO1 codon position was obtained by examining a prelimi- nary tree of mytilid relationships constructed in PAUP (without invoking differential weighting). The number of changes at each position was calculated using MacClade version 3.05 (Maddison and Maddison 1992). The relative difference in levels of sequence divergence between the CO1 and 16s RNA genes was calculated by taking the mean of the ratio between all pairwise p-distances for the CO1 and 16s RNA gene sequences as calculated in MEGA. Similarly, MEGA was used to calculate the TS/TV ratio for all pairwise comparisons among the M and F Myths mitotypes. The mean TS/TV ratio calculated from this matrix was then used to calculate the relative weight given to transversion-versus- transition substitutions as suggested by Hillis, Huelsen- beck, and Cunningham (1994).

We used the character optimization algorithm in MacClade on our best estimate of the relationships among all mytilid mitotypes to reconstruct the gender association of ancestral bivalve mitotypes. Essentially, we used the principle of parsimony to infer whether an ancestral mitotype was transmitted through a male or a female lineage. To facilitate the reconstruction of ancestral character states, we assumed that DUI evolved from standard maternal inheritance, which is presumably the ancestral condition in animals.

Results Phylogenetic Relationships

The weighted and unweighted MP and NJ trees generated from the inferred CO1 amino acid sequences (trees not shown) were similar to the topology presented in Hoeh et al. (1996) (fig. 1). That is, distinct gender- associated groupings of M and F mitotypes were indicated within the freshwater mussels and the marine mussel Geukensia demissa. These groupings were also supported in 100% of the bootstrapped replicates in the MP and NJ analyses. However, while a group consisting of the Myths sequences was supported in 100% of the bootstrapped trees, the branching order within this clade varied between the NJ and MP analyses. In all cases, however, the A4. californianus M type was basal to the remaining Mytilus sequences. To resolve relationships among the various M and F Mytilus sequences, we conducted two sets of additional analyses using (1) Geu- kensia as a functional outgroup (Maddison, Donoghue, and Maddison 1984), (2) nucleotide rather than amino acid sequences, and (3) the methods described above that compensate for among-site rate variation.

Using an unweighted parsimony analysis, we de- termined that there are approximately 3.7 and 20.4 times as many changes at first and third codon positions, respectively, as at second codon positions. This rank order of rates of change (i.e., r3 > rl > Y*) is commonly seen in protein-coding genes (Yang 1996). Three categories of rates, with a frequency of 0.333 each, were incor- porated into the DNAML analysis. To assign differential weights in PAUP, which requires whole numbers, we divided each of the above rates by 20.4 and took the inverse to obtain an approximate weighting scheme of 5, 20, and 1 for changes at first, second, and third positions, respectively. Similarly, because of a fourfold transition bias at first positions, transversions were assigned four times the weight of transitions for that codon position. Transitions and transversions occurred in roughly equal frequencies at second positions; therefore, no differential weighting was required. At third positions, however, transition substitutions were approaching saturation in some comparisons. To accommodate for this, we arbitrarily assigned third-position transversions 10 times the weight of third-position transitions to min- imize the potential impact of multiple transition substitutions at a site.

The matrix of pairwise evolutionary distances (estimated using the Tamura-Nei model with a gamma coefficient of 1.0) among all the mytilid CO1 sequences is

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Table 1 Evolutionary Distances (below diagonal, standard errors above diagonal) for All Pairwise Comparisons of Mytilid CO1 Sequences

F-ed M-A F-tr F-O M-O M-ed M-tr F-ca M-ca F-de M-de

F-ed. . . . 0.0073 0.0277 0.0283 0.0310 0.0399 0.033 1 0.0289 0.0366 0.0696 0.0742 M-A . . . 0.0277 0.0299 0.0310 0.033 1 0.0440 0.0342 0.0284 0.0367 0.0689 0.0765 F-tr . . . . 0.2050 0.2250 0.005 1 0.006 1 0.0391 0.03 11 0.0249 0.0376 0.0720 0.0686 F-O. . . . 0.2102 0.233 1 0.0150 0.0058 0.0405 0.0315 0.0258 0.0391 0.0722 0.070 1 M-O . . . 0.2276 0.2472 0.0203 0.0186 0.0404 0.0327 0.0273 0.0423 0.0749 0.0735 M-ed.. . 0.3135 0.3373 0.3 165 0.3276 0.3292 0.0388 0.0385 0.0501 0.0798 0.0896 M-tr. . . . 0.2730 0.283 1 0.2562 0.2608 0.2712 0.2930 0.0315 0.0411 0.0772 0.070 1 F-ca. . . . 0.2259 0.2197 0.1930 0.1987 0.2108 0.2964 0.263 1 0.0354 0.0600 0.0675 M-ca . . . 0.3256 0.3256 0.3353 0.347 1 0.3719 0.4368 0.3519 0.3115 0.0733 0.0688 F-de . . . 0.6353 0.6278 0.6438 0.6505 0.665 1 0.7199 0.6913 0.5627 0.665 1 0.0304 M-de... 0.6509 0.6547 0.6276 0.6412 0.6665 0.7730 0.6430 0.6174 0.6066 0.2556

NoTI%--Estimates are based on the Tamura-Nei model (Tamura and Nei 1993) (M-A and M-O) with the standard conspecific M and F sequences are underlined.

using a gamma coefficient of 1.0. Comparisons of newly masculinized types

presented in table 1. These distances were used in the NJ analysis.

For the 16s RNA nucleotide sequences used in the combined COI/16S RNA data set, transitions were three times more common than transversions. Accordingly, transversions were given three times the weight of transitions in the MP analysis. Similarly, since the 16s RNA gene was approximately two thirds as divergent as the CO1 sequences on average, we superimposed a 3:2 weighting scheme on the 16s RNA and CO1 portions of the combined data set. In the same manner, to compensate for differences in the rates of change of the two gene regions and differences among codon positions within COI, four categories of rates (2.0, 3.7, 1.0, and 20.4) were specified in the DNAML analysis of the combined data set. The value 2.0 was chosen because the level of divergence of a 16s RNA site is, on average, intermediate between first and second CO1 codon positions. The values 0.427, 0.191, 0.191, and 0.191 were also supplied to indicate the relative frequencies of 16s RNA sites and first, second, and third CO1 codon positions, respectively.

The phylogenetic trees resulting from the aforementioned analyses are shown in figures 2 (COI) and 3 (CO1 and 16s RNA). From the CO1 data alone, four features of the Mytilus phylogeny were invariable: (1) A4. californianus M was always a sister group to the remaining Mytilus sequences. This relationship was supported by bootstrap values of 91%-100%; (2) The male type M-A was always affiliated with the M. edulis F (bootstraps = 100%). (3) The M. trossulus M-O and F-O sequences formed a clade of their own which clus- tered with the standard M. trossulus F lineage (bootstraps L 99%). (4) The standard M types of M. edulis and M. trossulus were always grouped together (bootstrap values 1 83%). An important difference among the trees was the position of the M. californianus F type. Using MacClade, we calculated that the numbers of substitutions associated with each of these topologies (without invoking differential weighting of alternative character transformations) were 691 (MP), 690 (ML), and 689 (NJ). (The two most-parsimonious CO1 trees constructed without differential weighting [trees not shown] were 688 steps in length, and the position of the M.

californianus F type varied in each case.) Based on such small differences, it is difficult to prefer one of these topologies over the others.

Analysis of the combined COI/16S RNA data set helped resolve some of the aforementioned ambiguities. First, the ML, MP and NJ analyses all indicated that the M. edulis and A4. trossulus mitotypes (regardless of gender association) are descendants of a common ancestor shared with the M. californianus F mitotype (fig. 3). This set of relationships was supported by bootstrap values of 73%, 88%, and 91% in the MP, ML and NJ analyses, respectively. Relationships among the four remaining A4. edulis and A4. trossulus mitotypes varied slightly between the NJ and the MP and ML trees (the latter two trees being identical). The NJ trees both for the CO1 data alone (fig. 2C) and for the combined data set (fig. 3C) gave the division between M and F lineages of M. edulis and M. trossulus that has previously been proposed both by Stewart et al. (1995) based on CO111 gene sequences and by Rawson and Hilbish (1995) based on 16s RNA sequences. In contrast, the MP and ML trees suggested a clade consisting of the M. edulis F type as a sister group to a clade consisting of the A4. edulis and M. trossulus M types (fig. 3A and B). This portion of the topology was weakly supported, however, with bootstraps values of only 57%-62%. Given the above, the fully resolved NJ tree (fig. 2C) is regarded as the best estimate of mitotype relationships and, as such, will be used herein to interpret the evolutionary dynamics of DUI.

Reconstruction of Ancestral Gender Associations of Bivalve Mitotypes

Figure 4 is a synthesis of phylogenetic information from this study and Hoeh et al. (1996). Superimposed on this tree is the most parsimonious reconstruction of ancestral gender states as calculated in MacClade under the assumption that DUI (specifically, the generation and inheritance of M mitotypes) is a derived condition in bivalves, and therefore the ancestral gender state can be coded as E Masculinization events are therefore indicated at nodes 4, 5, and 6 and inferred to have taken place at nodes 1, 2, and 3.

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A

M. trossulus M A

100 M. trossulus F

100 Ir M. trossulus F-O

90 M. trossulus M-O

L M. trossulus F

L M. californianus F

M. californianus M

100 r -M. califomianus F

- M. californianus M I

7 G. demissa M

- G. demissa F rr M. edulis F 62

6

G. demissa F

r- M. edulis M

M. trossulus F-O

M. trossulus M-O M. trossulus M

96 ri L M. trossulus F LM. trossulus F

-M. californianus F

FM. edulis M L M. californianus F

F-- M. californianus M M. trossulus M

A G. demissa F

- M. edulis F C 65_

91 M. trossulus F

100 _

M. edulis M 96

M. trossulus M

M. trossulus M-O

M. trossulus F

C

100

.

-M. californianus F

M. californianus M

G. demissa F i, 011

FIG. 3.-The best estimates of the phylogenetic relationships among the mytilid M and F mitotypes obtained from analyzing 622 bp of CO1 and 458 bp of 16s RNA nucleotide sequences by three methods: (A) maximum parsimony, (B) maximum likelihood, and (C) neighbor-joining. Numbers indicate bootstrap support for each node. See text for details of the phylogenetic methodology. Geukensia demissa was used as the outgroup.

- M. trossulus M

-M. californianus F

M. californianus M

1-G. demissa M

FG. demissa F

0' 0.1

Discussion

Hoeh et al. (1996) recently demonstrated three distinct divergence events of M and F mitotypes in several distantly related bivalve species and proposed two ex- planations which might account for this pattern. If the formation of distinct M and F types is taken as an in- dication of a de novo initiation of DUI, then this complex phenomenon would have evolved at least three

FIG. 2.-The best estimates of the phylogenetic relationships among the mytilid M and F mitotypes obtained from analyzing 622 bp of CO1 sequence data by three methods: (A) maximum parsimony, (B) maximum likelihood, and (C) neighbor-joining. Numbers indicate bootstrap support for each node. See text for details of the phylogenetic methodology. Geukensia demissa was used as the outgroup.

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:““--- f. flava M : . . . . . . . . . . . f

li i___-- P. grandis M

i- P. fragilis M F. flava F P. grandis F

P. fragilis F

2 r G. demissa M

L G. demissa F ,_........................ Mm californianus M

31 M. californianus F

I-.. M. edulis M * . . . . . . . . . . . f I--- M. frossulus M

' 4: 5 !- M. edulis M-A

IF- M. edulis F

7 r M. rrossulus F

LL 6 y- M. trossulus M-O

M. trossulus F-O

FIG. 4.-Character optimization of ancestral mitotype gender associations inferred from the best estimates of phylogenetic relationships among mytilid mitotypes (NJ tree, fig. 2C) combined with the phylogenetic information of Hoeh et al. (1996) (fig. 1) and the assumption that DUI (i.e., the generation and inheritance of M mitotypes) is a derived condition in the Mollusca. Male-transmitted (M) mitotypes are shown in a dotted line. In addition, ancestral mitotypes inferred to be inherited via males are also indicated by a dotted line. Numbers indicate nodes on the tree where masculinization events (see text) are indicated (4, 5, and 6) or may have taken place (1, 2, and 3). (Note: branch lengths are not drawn to scale.)

times in the history of bivalves (fig. 1). This assumes that once established, the M and F types do not switch roles. In a review of DUI in mussels, Hurst and Hoekstra (1994) proposed that for such a system to remain stable, the M and/or F types must remain faithful to their own lineages. Alternatively, DUI may be a unique plesio- morphic trait in bivalves but reversals in the route of transmission of gender-associated types may occur pe- riodically. These hypothetical transitions were referred to as “masculinization” or “feminization” events by Hoeh et al. (1996). The strongest evidence in support of masculinization events comes from the phylogenetic af- finity of the M. trossulus type M-O and the M. e&&s type M-A with their respective conspecific F types (fig. 2). The derived position of these paternally transmitted mitochondrial genomes within M. edulis and M. tros-

sulus relative to the maternally transmitted mitotypes coupled with the close genetic similarity between these conspecific M and F types suggests that the paternally inherited mitotypes were recently spawned from female mitotypes (fig. 2 and table 1). Similarly, the phylogenetic position of the M. trossulus and M. edulis M types relative to the M. californianus F type (fig. 3) also indicates another instance of masculinization of female mtDNA, assuming that this is the true phylogeny of these genomes (see below). By extension, we propose that the same process could have occurred repeatedly in the history of the bivalves. This hypothesis is summa- rized in figure 4, which is a synthesis of phylogenetic information from this study and Hoeh et al. (1996) combined with the most parsimonious reconstruction of ancestral gender states. Transitions 4, 5, and 6 are only

compatible with masculinization events. Under the assumption that DUI arose once in an ancestral bivalve lineage (prior to node A, fig. 4), additional masculinization events must have occurred at position 1 within the Unionidae and at positions 2 and 3 within the My- tilidae.

As previously described (Rawson and Hilbish 1995; Stewart et al. 1995, 1996), the “standard” M mitotypes in A4. edulis and M. trossulus arose from an M mitotype which existed in the common ancestor of these two species. In contrast, it may be inferred from the comparatively small genetic divergences between the M-O and M-A mitotypes and their respective conspecific F types (table 1) that the former two mitotypes arose relatively recently. Furthermore, these newly masculinized M mitotypes are coexisting with the “old” M mitotypes in a state of polymorphism within these species. M-O occurs at a frequency of 0.24 in M. trossulus males and M-A occurs at a frequency of 0.26 in A4. edulis males sampled from Lunenburg Bay, Nova Scotia (Stewart et al. 1995). By comparison, only one class of male types has been found in the other species so far. This is not surprising given that only species of the M. edulis complex (i.e., M. edulis, M. trossulus, and M. galloprovincialis) have been examined in numbers large enough to reveal the existence of polymorphism. How- ever, the fact that the mitotypes from two unionid genera affiliate according to gender, whereas those from two genera of the Mytilidae (i.e., Mytilus and Geukensia) do not, poses the question of whether there are taxonomic differences in the stability of DUI. As stated by Hoeh et al. (1996), the time of the split between M and F mitotypes in the freshwater mussels (and therefore the length of time for which DUI has operated in a stable fashion) may be much longer than the time indicated for the Mytilidae. Given that the taxonomic split between the Pyganodon and Fusconaia lineages occurred at least 100 MYA, Hoeh et al. (1996) deduced that the M and F mitotypes in these species are at least 100 Myr old. In contrast, Rawson and Hilbish (1995) estimated the time of the split between the most common M and F mitotypes in the M. edulis species complex at 5.3 MYA. Hoeh et al. (1996) estimated that the formation of the M and F mitotypes in Geukensia was of a similar age. The newly masculinized M mitotypes in Mytilus are clearly of even more recent origin. In contrast to the large divergences between the standard conspecific M and F mitotypes in M. edulis and A4. trossulus (ca. 25%- 30%), the newly masculinized mitotypes have diverged from their conspecific female types by less than 2.8% (table 1).

The sequence of events that led to masculinization remains unknown. However, one possible mechanism is supported by recent studies of mtDNA transmission in Mytilus (Zouros et al. 1994b; Saavedra, Reynero, and Zouros 1997). In these crosses, some males failed to pass either the M or F mitotype to their sons. A small percentage of sons from other males also failed to inherit mtDNA from their fathers. The overall rate of M-negative males in these crosses was about 20%. Although the information existing so far is limited to a few cases,

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M-negative males produce sperm that contains the mtDNA of the mother (Saavedra, Reynero, and Zouros 1997). Thus, there is direct evidence that in the absence of an M type in a male, the F molecule will assume the role of the M molecule in sperm. It is not yet known, however, if M-negative males are actually fertile or, if so, whether they produce sons.

Although Hoeh et al. (1996) proposed that role reversals could theoretically occur in either direction, it is important to note that the demonstrated reversals in routes of transmission in the genus Myth as well as the possible reversals in the remaining bivalve taxa are all compatible with masculinization events. However, our hypothesis that only changes from F to M occur must be qualified given the difficulties of phylogenetic reconstruction among sequences with such variable rates of character transformations (Huelsenbeck and Hillis 1993; Hillis, Huelsenbeck, and Cunningham 1994). Nonetheless, other interpretations of Mytilus mitotype relationships do not appear to be plausible. For instance, if the standard M types of M. edulis and M. trossulus are placed in a clade with the A4. californianus M mitotype (thereby eliminating one masculinization event), the length of the tree increases considerably (from 689 to 710 steps). Furthermore, to accept an alternate topology such as either of those presented in figure 2A or B, we must invoke a relatively more complicated biological explanation that incorporates some form of hybridiza- tion and introgression of mitochondrial DNA across species boundaries to account for the pattern of mitotype relationships. Based on the results of a survey of 201 “pure” and “hybrid” M. edulis and M. trossulus mussels from the wild, Saavedra et al. (1996) concluded that there are intrinsic barriers to the exchange of mtDNA between these two species. The bias in favor of masculinization as opposed to feminization events is also consistent with the data currently available concerning within-individual mitotype distributions in Mytilus. The number of M-positive females is much smaller than that of M-negative males, and the amount of M mitotype in the former is very small, whereas in the latter the F molecule is 100%. Furthermore, there is, as yet, no evidence of eggs containing M molecules but, as noted, there is evidence that M-negative males produce sperm that contains the F molecule (Saavedra, Reynero, and Zouros 1997). While alternate interpretations invoking feminization events do not appear to be in accord with our limited data from the wild or from laboratory crosses, they cannot, for the moment, be completely discount- ed.

Role reversal of an F mitotype to yield a newly masculinized M mitotype is presumably the first step in a process which resets, initially to zero, the amount of sequence divergence (and apparent time since divergence) between a species’ M and F mitotypes. The second step is a transient state of polymorphism in which both new and old M mitotypes coexist in the population. The third step is replacement of the old M mitotype by a newly masculinized type (see Hoeh et al. 1996, fig. 3). We can illustrate how these stages would have pro- ceeded for the species of the genus Mytilus (fig. 5). First,

M. calijomianus

M F

M. edulis

M F M-A

M. trossulus

M M-O F

Taxon

FIG. 5.-Hypothetical reconstruction of speciation, mitotype masculinization, and mitotype extinction events which could explain the observed phylogenetic relationships of the M and F mitotypes sampled in extant members of the genus A4yriZus. The heavy solid lines outline organismal phylogeny. The light solid lines represent F mitotype phylogeny while the dashed lines represent M mitotype phylogeny. Chro- nology: A, An ancestral Mytilus lineage displays DUI. B, Allopatric speciation occurs such that an ancestral species bifurcates to produce two descendant lineages. One of these leads to the M. edulis/M. cros- sulus complex and the other to M. californianus. C, In the line leading to the M. edulislhf trossulus complex and before the separation of h4. edulis and M. trossulus, the F mitotype lineage spawned an M mitotype via a masculinization event. D, The common ancestor of 44. edulis and M. trossulus would then have passed through a transitional state of polymorphism for the newly masculinized M mitotype and the preexisting M mitotype. E, The original M mitotype in the M. edulis/M. trossulus complex went on to extinction. F, Allopatric speciation leading to M. edulis and M. trossulus. G, Masculinization event giving rise to the M. edufis M-A mitotype. H, Masculinization event giving rise to the M. trossulus M-O mitotype. The M. trossulus F-O mitotype has been omitted to simplify the presentation.

assume a standard mode of allopatric speciation in which an ancestral DUI-containing species (fig. 5A) bifurcates to produce two descendant lineages (fig. 5B). One of these leads to the M. eduZis/M. trossulus complex, and the other leads to M. californianus. Presum- ably, each of these descendant lineages contained ho- mologous (orthologous) M and F mitotypes. In the line leading to the M. eduZislA4. trossulus complex and before the separation of M. edulis and M. trossulus, the F lineage spawned an M mitotype via a masculinization event (fig. 5c). The common ancestor of M. edulis and M. trossulus would then have passed through a transi-

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966 Hoeh et al.

tional state of polymorphism for the newly masculinized M mitotype and the preexisting M mitotype (fig. 5D). Finally, the original M mitotype presumably went on to extinction (fig. 5E), since we have not, as yet, found an M mitotype in the M. eduZislA4. trossulus complex that is a sister mitotype to the A4. californianus M mitotype. As mentioned above, a consequence of this process is that the apparent origin of DUI (i.e., divergence of F and M mitotypes) in the A4. eduZis/M. trossulus species complex is moved forward in time to the point of the most recent masculinization event.

From a population genetics point of view, we want to know if the replacement of the old M lineage by a newly masculinized lineage is a stochastic event, or if the latter has intrinsic advantages over the former. It is well established that the M lineage evolves faster than the F lineage, and we have presented evidence that the reason for this is relaxed selection against the M type (Stewart et al. 1996). This mechanism might favor the accumulation of slightly deleterious mutations which would predispose the old M type to extinction under competition with a new M type. On the other hand, this could lead to very frequent masculinization events. This is clearly not the case in freshwater mussels. We also note that if masculinizations occur very often and if newly spawned molecules quickly replace the old molecules, this would not allow for the mutational divergence of M and F molecules. In such systems, the existence of DUI could remain undetected.

Acknowledgments

We thank the following for assistance in the pro- curement and/or maintenance of specimens: W. Borge- son, D. Cook, D. Hedgecock, C. Herbinger, E. Ken- chington, J. Maunder, R. Noseworthy, and R. Trdan. A. Ball, S. Baldauf, D. Cook, and M. Dillon kindly provided assistance with laboratory procedures and (or) provided access to the facilities of the Marine Gene Probe Laboratory, Dalhousie University. We also thank Andrew Martin and an anonymous reviewer for numer- ous constructive comments on this manuscript. This research was supported by grants from the Natural Sci- ences and Engineering Research Council of Canada (NSERC) to E.Z. and by the Research Development Fund (Dalhousie University) to W.R.H. and D.T.S. W.R.H. and D.T.S. were supported by postdoctoral fellowships from NSERC. C.S. was supported by postdoctoral fellowships from the Conselleria de Education, Xunta de Galicia (Spain) and the Ministerio de Educa- cion y Ciencia (Spain).

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CARO-BETH STEWART, reviewing editor

Accepted May 30, 1997

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phylogenetic evidence for role-reversals of gender-associated

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