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Mol Biol Evol-1997-Hoeh-959-67

VIEWS: 9 PAGES: 9

									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                   the F sequences and the M sequences from all three
(DUI) of mitochondrial        DNA (mtDNA) has now been                          species formed two gender-associated       groups. The same
documented       in several phylogenetically    diverse bivalve                 result was observed by Stewart et al. (1995) in a col-
taxa. These taxa include marine mussels of the family                           lection of cytochrome c oxidase subunit III (COIII) se-
Mytilidae     (Myti2u.s edu2is [Skibinski,      Gallagher,   and                quences from M. edulis and M. trossulus. Both studies
Beynon 1994a, 1994b; Zouros et al. 1994a, 1994b], M.                            concluded that the formation of distinct F and M lin-
trossulus [Geller 1994; Zouros et al. 1994b; Rawson and                         eages must have preceded the emergence of the three
Hilbish 1995; Stewart et al. 19951, M. galloprovincialis                        species of mussels from their common ancestor.
[Rawson and Hilbish 1995; Quesada, Skibinski,                and                       The discovery of DUI in freshwater mussels (Liu,
Skibinski 1996; Saavedra, Reynero, and Zouros 19971,                            Mitton, and Wu 1996) prompted Hoeh et al. (1996) to
Geukensia demissa [Hoeh et al. 19961) and freshwater                            examine the phylogeny of M and F mitotypes in both
mussels of the family Unionidae (Pyganodon fragilis, P.                         marine and freshwater mussels. Specifically, these au-
grandis and Fusconaia Jlava [Liu, Mitton, and Wu 1996;                          thors compared CO1 sequences from six taxa: M. edulis,
Hoeh et al. 19961). In these bivalves, males possess two                        M. trossulus, G. demissa, P. grandis, P. fragilis, and F.
distinct classes of mtDNA: an M type, so-named be-                              Java. As expected from the previous analysis of 16s
cause it is transmitted from male parents to their sons,                        RNA (Rawson and Hilbish 1995) and CO111 sequences
and an F type that is transmitted           from generation    to                (Stewart et al. 1995), Hoeh et al. (1996) obtained dis-
generation through females. Although sons also receive                          tinct M and F groupings for the Myths         mitotypes (fig.
               s
their mother’ F type mtDNA, they do not transmit it to                           1). The six M and F sequences of the three unionid
their offspring.                                                                 species (P. grandis, P. fragilis, and F. Java) also formed
       Rawson and Hilbish (1995) and Stewart et al.                             two distinct groups defined on the basis of gender, but
( 1995) were the first to examine DUI from a phyloge-                           as a whole, the freshwater mussel sequences formed a
netic perspective.     Using mitochondrial      16s RNA gene                    group that was distinct from the marine mussel sequenc-
sequences from M. edulis, M. trossulus, and A4. gallo-                          es. The M and F types present in Geukensia formed a
provincialis,     Rawson and Hilbish (1995) showed that                          sister group to the other mytilid sequences. The com-
                                                                                plete phylogenetic     tree of the 12 sequences indicated at
     I Present address: Department       of Zoology,   Miami University.
                                                                                least three separate divergences      of M and F mitotype
                                                                                lineages, one in each of the lines leading to Myths       and
     2 Present   address:   Department   of Biology,   University   of Crete.
                                                                                to Geukensia and one in the line leading to freshwater
     3 Present address: Department       of Microbiology    and Immunology,
                                                                                 mussels (fig. 1). Hoeh et al. (1996) inferred from these
University of British Columbia.
                                                                                three F/M mitotype divergence events either that there
      Key words: phylogenetics,  mitochondrial  DNA, doubly unipar-
                                                                                have been multiple origins of DUI in bivalves or that
ental inheritance, cytochrome c oxidase I, 16s RNA, masculinization,
Mytilus, Bivalvia, Mytilidae.
                                                                                there was a single origin of DUI in the ancestral bivalve
                                                                                 lineage but reversals in the route of mitotype transmis-
     Address for correspondence  and reprints: Donald T. Stewart, De-
partment of Biology, Fairfield University, Fairfield, CT 06430-5 195.            sion had mimicked de novo divergences          of F and M
E-mail: dstewart@is.dal.ca.                                                      lineages. Specifically, Hoeh et al. (1996) referred to the
Mol. Bid. Evol. 14(9):959-967. 1997
                                                                                hypothetical    spawning of an M type from an F type as
0 1997 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038       a “masculinization”       event. The corresponding    switch

                                                                                                                                             959
960    Hoeh et al.


                                     r       F. flava M
                                                                              quencing Kit, Perkin Elmer) or by direct sequencing

                                3        L
                                             P. grandis M

                                             P. fragilis M
                                                                              (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.
                                             F. flava F                      Consequently,      85%-90% of the total sequence obtained
                                             P. grandis F                    was confirmed by sequencing in both directions.
                                                                                    We have also obtained additional CO1 sequences of
                                -k           P. fragilis F
                                                                             two M. trossulus mitotypes and one M. edulis mitotype
                                         r   G. demissa M                     (designated    M-O, F-O, and M-A, respectively).       M-O
                                                                             and M-A were classified as M types because they were
                     1         /L.              damissa F
                                                                             isolated from the gonads of males (Saavedra, Reynero,
                                             M. edulis M                     and Zouros 1996). Restriction mapping (data not shown)




                     -2
      FIG. l.-Topology
                                     f
                                             M. trossulus

                                             M. edulis F

                                             M. trossulus
                                                             M



                                                             F
                            from Hoeh et al. (1996) indicating three di-
vergence events between M and F mitotypes (shown by arrows) in a
                                                                             indicated that these putative mitochondrial
                                                                             were of a size consistent with mitochondrial
                                                                                                                              DNA types
                                                                                                                                 genomes
                                                                             and therefore were not the result of transfer of mito-
                                                                             chondrial 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
collection of freshwater and marine mussels. The tree suggests that one      of M. edulis and M. trossulus, respectively, these mito-
divergence event occurred in an ancestor to the freshwater mussel spe-       types were not previously sequenced because of tech-
cies P. grandis, P. fragilis, and F. Jlava, another in the lineage leading
                                                                             nical difficulties (see Stewart et al. 1995). Briefly, be-
to the marine mussel G. demissa, and one in a common ancestor to
M. edulis and M. trossulus.                                                  cause 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 diffi-
from an M to an F type would be referred to as a “fem-                       cult to isolate the CO111 amplification    products of M-O
inization”    event. This hypothesis was based on obser-                     and M-A from the amplification        products of their ac-
vations that the fidelity of DUI is not always perfect.                      companying      F types. The newly obtained CO1 sequenc-
That is, females containing an M type and males lacking                      es were manually aligned, using MacClade (Maddison
an M type have occasionally        been observed (Fisher and                 and Maddison        1992), with CO1 sequences previously
Skibinski     1990; Zouros et al. 1992, 1994b; Saavedra,                     analyzed by Hoeh et al. (1996).
Reynero, and Zouros 1997). Such anomalous individuals                               We also amplified and obtained sequence from a
could provide information        about how an mtDNA type                     527-bp portion of the mitochondrial         16s RNA gene
associated with otie gender lineage could switch its route                   from two male and two female M. californianus speci-
of transmission     to the opposite gender lineage.                          mens using the primer pair 16s AR and 16s BR (Pal-
       To further evaluate the alternate hypotheses indi-                    umbi et al. 199 1; Rawson and Hilbish 1995). For an
cated above, we have characterized        additional mitotype                outgroup, we sequenced the F type from two female
lineages within Mytilus for CO1 and 16s RNA and phy-                         Geukensia demissa. The amplification protocol followed
logenetically    analyzed these sequences in conjunction                     Rawson and Hilbish (1995) and the product was se-
with the CO1 sequences presented in Hoeh et al. (1996)                       quenced directly as described above. These sequences
and the 16s RNA sequences presented in Rawson and                            were aligned against 458 bp of 16s RNA sequence of
Hilbish (1995). From these analyses, we present evi-                         A4. edulis and M. trossulus M and F types extracted from
dence that M mitotypes in the genus Mytilus are derived                      the GenBank database (U22864, U22865, U22879, and
from multiple ancestral lineages. We then demonstrate                        U22882; Rawson and Hilbish 1995).
how the reversal of the transmission      route of F genomes                        The CO1 and 16s RNA sequences generated herein
(i.e., masculinization    events) best accounts for the ob-                  have been submitted to the GenBank database (acces-
served phylogenetic      pattern, and we suggest a sequence                  sion numbers         U68770-U68772       [16S RNA]       and
of events to explain the groupings of M and F types at                       U68773-U68777         [COI]).
different levels of the bivalve mitochondrial       phylogeny.
                                                                             Phylogenetic   Reconstruction

Materials and Methods                                                               In this paper, we present three sets of phylogenetic
Sequencing Protocol                                                          reconstructions.    The first set was conducted to test the
                                                                             validity of the deeper nodes of our previously reported
     Total DNA was isolated from either gonad or                             topology (see Hoeh et al. 1996, fig. 1). To this end, we
spawned gametes of both sexes of Mytilus californianus.                      conducted     maximum-parsimony       (MP) (PAUP version
This material was used to amplify a 710-bp fragment of                       3.1.1; Swofford       1993) and neighbor-joining        (NJ)
the mitochondrial  cytochrome   oxidase subunit I gene                       (MEGA version 1.02; Kumar, Tamura, and Nei 1993)
(COI) using the primers LCO1490 and HC02198          (for                    analyses on the inferred amino acid sequences of our
primer sequences and details of the amplification   pro-                     CO1 data set (i.e., 11 mytilid and 6 unionid sequences).
tocol see Folmer et al. [1994] and Hoeh et al. [ 19961).                     The MP analysis was conducted with and without in-
These primers were also used to generate 622 bp of CO1                       voking a step matrix to weight amino acid substitutions
sequence either by cycle sequencing    (AmpliCycle   Se-                     relative to the minimum number of changes required to
                                                                                               Masculinization   of mtDNA   in Mytilus   961


 switch from one amino acid to another. NJ trees were                       We used the character optimization        algorithm in
 constructed    using an uncorrected        amino acid distance       MacClade      on our best estimate of the relationships
 matrix and matrices for which distances were estimated               among all mytilid mitotypes to reconstruct the gender
 using gamma parameters           (a) of 0.5, 1.0, and 2.0, re-       association of ancestral bivalve mitotypes. Essentially,
 spectively.                                                          we used the principle of parsimony to infer whether an
        In the second set of analyses, we focus on the re-            ancestral mitotype was transmitted through a male or a
 lationship among the nine M and F Mytilus sequences                  female lineage. To facilitate the reconstruction    of ances-
 using the CO1 nucleotide data. The methods used in-                  tral character states, we assumed that DUI evolved from
cluded       MP, NJ and maximum               likelihood      (ML)    standard maternal inheritance, which is presumably the
 (DNAML, PHYLIP version 3.5~; Felsenstein 1993). The                  ancestral condition in animals.
ML analysis was performed using 100 random terminal
mitotype addition sequence runs with the global rear-                 Results
rangement option in force. The third set of analyses was              Phylogenetic    Relationships
conducted      on a subset of the mytilid sequences for
which a combined CO1 and 16s RNA nucleotide data                             The weighted and unweighted          MP and NJ trees
set was available. Although the CO1 and 16s RNA se-                   generated from the inferred CO1 amino acid sequences
quences for a given mitotype (e.g., M. trossulus M) were              (trees not shown) were similar to the topology presented
not obtained from the same individual, it is assumed that             in Hoeh et al. (1996) (fig. 1). That is, distinct gender-
the two sequences that were pooled to form a composite                associated groupings of M and F mitotypes were indi-
mitotype share a more recent common ancestor with                     cated within the freshwater mussels and the marine mus-
each other than they do with any other sequence includ-               sel Geukensia demissa. These groupings were also sup-
ed in the analysis. Again, all three methods of tree re-              ported in 100% of the bootstrapped replicates in the MP
construction     were used. Percent bootstrap support for             and NJ analyses. However, while a group consisting of
the resulting nodes was evaluated using 1,000 replica-                the Myths       sequences was supported in 100% of the
tions for all of the MP and NJ analyses and 100 repli-                bootstrapped trees, the branching order within this clade
cations for the ML analysis (Hillis and Bull 1993).                   varied between the NJ and MP analyses. In all cases,
       In the second set of analyses, we used methods that            however, the A4. californianus M type was basal to the
take into consideration        different rates of sequence di-        remaining    Mytilus sequences. To resolve relationships
vergence among sites and different transition (TS) and                among the various M and F Mytilus sequences, we con-
transversion     (TV) ratios across codon positions within            ducted two sets of additional analyses using (1) Geu-
the CO1 gene. The ML and NJ analyses also corrected                   kensia as a functional outgroup (Maddison, Donoghue,
for differences      in frequencies     of the four nucleotides.      and Maddison 1984), (2) nucleotide rather than amino
Specifically,    the NJ tree was reconstructed         from a dis-    acid sequences, and (3) the methods described above
tance matrix estimated using the Tamura-Nei model (Ta-                that compensate for among-site rate variation.
mura and Nei 1993) and a gamma coefficient of 1 .O. In                       Using an unweighted       parsimony analysis, we de-
the third set of analyses, we were also able to compen-               termined that there are approximately        3.7 and 20.4 times
sate for the difference in the overall level of sequence              as many changes at first and third codon positions, re-
divergence between the CO1 and 16s RNA genes in the                   spectively, as at second codon positions. This rank order
MP and ML analyses. These weighting schemes were                      of rates of change (i.e., r3 > rl > Y*) is commonly seen
implemented       to give more weight to presumably            more   in protein-coding     genes (Yang 1996). Three categories
conservative      changes (Hillis, Huelsenbeck,          and Cun-     of rates, with a frequency of 0.333 each, were incor-
ningham 1994) and/or to estimate, as accurately as pos-               porated into the DNAML analysis. To assign differential
sible, the evolutionary      distance between mitotypes (Ku-          weights in PAUP, which requires whole numbers, we
mar, Tamura, and Nei 1993).                                           divided each of the above rates by 20.4 and took the
       The approximate       number of changes at each CO1            inverse to obtain an approximate weighting scheme of
codon position was obtained by examining                 a prelimi-   5, 20, and 1 for changes at first, second, and third po-
nary tree of mytilid relationships         constructed in PAUP        sitions, respectively.    Similarly, because of a fourfold
(without invoking differential         weighting).    The number      transition bias at first positions, transversions        were as-
of changes        at each position        was calculated      using   signed four times the weight of transitions for that codon
MacClade version 3.05 (Maddison and Maddison 1992).                   position.   Transitions     and transversions        occurred     in
The relative difference in levels of sequence divergence              roughly equal frequencies at second positions; therefore,
between the CO1 and 16s RNA genes was calculated                      no differential    weighting was required. At third posi-
by taking the mean of the ratio between all pairwise                  tions, however, transition substitutions were approaching
p-distances    for the CO1 and 16s RNA gene sequences                 saturation in some comparisons.         To accommodate          for
as calculated in MEGA. Similarly, MEGA was used to                    this, we arbitrarily assigned third-position        transversions
calculate the TS/TV ratio for all pairwise comparisons                 10 times the weight of third-position      transitions to min-
among the M and F Myths            mitotypes. The mean TS/TV          imize the potential impact of multiple transition substi-
ratio calculated from this matrix was then used to cal-               tutions at a site.
culate the relative weight given to transversion-versus-                     The matrix of pairwise evolutionary         distances (es-
transition substitutions     as suggested by Hillis, Huelsen-         timated using the Tamura-Nei model with a gamma co-
beck, and Cunningham          (1994).                                 efficient of 1.0) among all the mytilid CO1 sequences is
962     Hoeh et al.


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) using a gamma coefficient   of 1.0. Comparisons   of newly masculinized    types
(M-A and M-O) with the standard conspecific M and F sequences are underlined.


presented in table 1. These distances were used in the                      californianus F type varied in each case.) Based on such
NJ analysis.                                                                small differences,    it is difficult to prefer one of these
        For the 16s RNA nucleotide sequences used in the                    topologies over the others.
combined COI/16S RNA data set, transitions were three                              Analysis of the combined COI/16S RNA data set
times more common than transversions.           Accordingly,                helped resolve some of the aforementioned        ambiguities.
transversions    were given three times the weight of tran-                 First, the ML, MP and NJ analyses all indicated that the
sitions in the MP analysis. Similarly, since the 16s RNA                    M. edulis and A4. trossulus mitotypes (regardless of gen-
gene was approximately       two thirds as divergent as the                 der association) are descendants of a common ancestor
CO1 sequences        on average, we superimposed        a 3:2               shared with the M. californianus F mitotype (fig. 3).
weighting scheme on the 16s RNA and CO1 portions                            This set of relationships was supported by bootstrap val-
of the combined data set. In the same manner, to com-                       ues of 73%, 88%, and 91% in the MP, ML and NJ anal-
pensate for differences in the rates of change of the two                   yses, respectively.     Relationships   among the four re-
gene regions and differences         among codon positions                  maining A4. edulis and A4. trossulus mitotypes varied
within COI, four categories of rates (2.0, 3.7, 1.0, and                    slightly between the NJ and the MP and ML trees (the
20.4) were specified in the DNAML analysis of the                           latter two trees being identical). The NJ trees both for
combined data set. The value 2.0 was chosen because                         the CO1 data alone (fig. 2C) and for the combined data
the level of divergence of a 16s RNA site is, on average,                   set (fig. 3C) gave the division between M and F lineages
intermediate    between first and second CO1 codon posi-                    of M. edulis and M. trossulus that has previously been
tions. The values 0.427, 0.191, 0.191, and 0.191 were                       proposed both by Stewart et al. (1995) based on CO111
also supplied to indicate the relative frequencies of 16s                   gene sequences      and by Rawson and Hilbish (1995)
RNA sites and first, second, and third CO1 codon po-                        based on 16s RNA sequences. In contrast, the MP and
sitions, respectively.                                                      ML trees suggested a clade consisting of the M. edulis
       The phylogenetic    trees resulting from the afore-                  F type as a sister group to a clade consisting of the A4.
mentioned analyses are shown in figures 2 (COI) and 3                       edulis and M. trossulus M types (fig. 3A and B). This
(CO1 and 16s RNA). From the CO1 data alone, four
                                                                            portion of the topology was weakly supported, however,
features of the Mytilus phylogeny were invariable:         (1)
                                                                            with bootstraps values of only 57%-62%.           Given the
A4. californianus M was always a sister group to the
                                                                            above, the fully resolved NJ tree (fig. 2C) is regarded
remaining Mytilus sequences. This relationship was sup-
                                                                            as the best estimate of mitotype relationships        and, as
ported by bootstrap values of 91%-100%;         (2) The male
                                                                            such, will be used herein to interpret the evolutionary
type M-A was always affiliated with the M. edulis F
                                                                            dynamics of DUI.
(bootstraps    = 100%). (3) The M. trossulus M-O and
F-O sequences formed a clade of their own which clus-                       Reconstruction  of Ancestral           Gender      Associations       of
tered with the standard M. trossulus F lineage (boot-                       Bivalve Mitotypes
straps L 99%). (4) The standard M types of M. edulis
and M. trossulus were always grouped together (boot-                               Figure 4 is a synthesis of phylogenetic information
strap values 1 83%). An important difference among                          from this study and Hoeh et al. (1996). Superimposed
the trees was the position of the M. californianus F type.                  on this tree is the most parsimonious      reconstruction  of
Using MacClade, we calculated that the numbers of sub-                      ancestral gender states as calculated in MacClade under
stitutions associated with each of these topologies (with-                  the assumption that DUI (specifically, the generation and
out invoking differential     weighting of alternative char-                inheritance   of M mitotypes) is a derived condition in
acter transformations)    were 691 (MP), 690 (ML), and                      bivalves, and therefore the ancestral gender state can be
689 (NJ). (The two most-parsimonious        CO1 trees con-                  coded as E Masculinization       events are therefore indi-
structed without differential weighting [trees not shown]                   cated at nodes 4, 5, and 6 and inferred to have taken
were 688 steps in length, and the position of the M.                        place at nodes 1, 2, and 3.
                                                                                                               Masculinization         of mtDNA in Mytifus   963



           A                                                                                  A
                                                               M. trossulus M




                              100




             Ir
                                                     M. trossulus F                                                      L M. trossulus F
                                           100




           r
                                                      M. trossulus F-O
                   100
                                                                                                                L            M. californianus F
                                      90               M. trossulus M-O
                                                                                                               M. californianus M
                                           -M.             califomianus    F

                          -            M. californianus M                                 I                                             G. demissa F

                                                 7             G. demissa M




                                                                                                                             rr
                                                                                                                                   M. edulis F
                                                 -             G. demissa F                                               62
                                                                                              6

                                                                                                                                        M. edulis M
                                                            M. trossulus F-O                                                      r-



                                                            M. trossulus M-O                                                            M. trossulus M




                                                 -M.
                                                      LM. trossulus F

                                                           californianus   F                         ri    96




                                                                                                                     L
                                                                                                                             L M. trossulus F


                                                                                                                              M. californianus       F
                                                 FM.           edulis M

                                                          M. trossulus M
                                                                                          F--
                                                                                          A
                                                                                                                         M. californianus        M


                                                                                                                                       G. demissa F


                                                                                                                         -         M. edulis F
                                                                                               C                    65_

                                                                                                                91
                                                                                                                                  M. trossulus F
                                                                                                         100    _

                                                                                                                                          M. edulis M
                                                                                                                     96


                                                                                                                                        M. trossulus M

               C                                        M. trossulus M-O                                         -M.              californianus      F
                                                       M. trossulus F
                                                                                                                                M. californianus         M


                                                                                                                                           G. demissa F
                                                                                              i,   011

                   100
                                                                                     FIG. 3.-The     best estimates of the phylogenetic   relationships
                                                                                among the mytilid M and F mitotypes obtained from analyzing 622
                                       -                  M. trossulus M        bp of CO1 and 458 bp of 16s RNA nucleotide sequences by three
                                -M.                    californianus   F        methods: (A) maximum parsimony, (B) maximum likelihood, and (C)
                                                                                neighbor-joining.   Numbers indicate bootstrap support for each node.
                                                       M. californianus    M    See text for details of the phylogenetic  methodology.  Geukensia de-
                                                                                missa was used as the outgroup.

          .                                1-G.                demissa M

          FG.                                                  demissa F        Discussion
                     0'       0.1
                                                                                       Hoeh et al. (1996) recently demonstrated three dis-
      FIG. 2.-The    best estimates of the phylogenetic      relationships
                                                                                tinct divergence events of M and F mitotypes in several
among the mytilid M and F mitotypes obtained from analyzing 622
bp of CO1 sequence data by three methods: (A) maximum parsimony,
                                                                                distantly related bivalve species and proposed two ex-
(B) maximum likelihood, and (C) neighbor-joining.      Numbers indicate         planations which might account for this pattern. If the
bootstrap support for each node. See text for details of the phylogenetic       formation of distinct M and F types is taken as an in-
methodology.    Geukensia demissa was used as the outgroup.                     dication of a de novo initiation of DUI, then this com-
                                                                                plex phenomenon       would have evolved at least three
964   Hoeh et al.


                                              “
                                            :“ --- f. flava M                 compatible with masculinization       events. Under the as-
                               : ...........f
                                                   P.
                                            i___-- grandis M                  sumption that DUI arose once in an ancestral bivalve
                              li             i-   P. fragilis M               lineage (prior to node A, fig. 4), additional masculini-
                                                  F. flava F                  zation events must have occurred at position 1 within
                                                  P. grandis F
                                                                              the Unionidae and at positions 2 and 3 within the My-
                                                  P. fragilis F
                                                                              tilidae.
                                         2 r G. demissa M                            As previously     described  (Rawson       and Hilbish
                                            L G. demissa F                    1995; Stewart et al. 1995, 1996), the “standard”        M mi-
                           ,_........................ californianus
                                                 Mm                   M       totypes in A4. edulis and M. trossulus arose from an M
                         31                       M. californianus    F       mitotype which existed in the common ancestor of these
                                             I-.. M. edulis M
                                                                              two species. In contrast, it may be inferred from the
                                    *...........f                             comparatively     small genetic divergences       between the
                                                I--- frossulus M
                                                   M.
                                                                              M-O and M-A mitotypes and their respective conspe-
                                   4:'   5 !-     M. edulis M-A
                                                                              cific F types (table 1) that the former two mitotypes
                                   IF-            M. edulis F                 arose relatively recently. Furthermore, these newly mas-
                                     7   r        M. rrossulus F              culinized M mitotypes are coexisting with the “old” M

                                         LL
                                         6 y- M. trossulus M-O
                                                  M. trossulus F-O
                                                                              mitotypes in a state of polymorphism        within these spe-
                                                                              cies. 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.
      FIG. 4.-Character     optimization of ancestral mitotype gender as-
sociations inferred from the best estimates of phylogenetic       relation-   edulis males sampled from Lunenburg Bay, Nova Scotia
ships among mytilid mitotypes (NJ tree, fig. 2C) combined with the            (Stewart et al. 1995). By comparison, only one class of
phylogenetic    information   of Hoeh et al. (1996) (fig. 1) and the as-      male types has been found in the other species so far.
sumption that DUI (i.e., the generation and inheritance of M mitotypes)       This is not surprising given that only species of the M.
is a derived condition in the Mollusca. Male-transmitted     (M) mitotypes
are shown in a dotted line. In addition, ancestral mitotypes inferred to
                                                                              edulis complex (i.e., M. edulis, M. trossulus, and M.
be inherited via males are also indicated by a dotted line. Numbers           galloprovincialis) have been examined in numbers large
indicate nodes on the tree where masculinization      events (see text) are   enough to reveal the existence of polymorphism.          How-
indicated (4, 5, and 6) or may have taken place (1, 2, and 3). (Note:         ever, the fact that the mitotypes from two unionid genera
branch lengths are not drawn to scale.)
                                                                              affiliate according to gender, whereas those from two
                                                                              genera of the Mytilidae (i.e., Mytilus and Geukensia) do
times in the history of bivalves (fig. 1). This assumes                       not, poses the question of whether there are taxonomic
that once established, the M and F types do not switch                        differences in the stability of DUI. As stated by Hoeh
roles. In a review of DUI in mussels, Hurst and Hoekstra                      et al. (1996), the time of the split between M and F
(1994) proposed that for such a system to remain stable,                      mitotypes in the freshwater mussels (and therefore the
the M and/or F types must remain faithful to their own                        length of time for which DUI has operated in a stable
lineages. Alternatively,    DUI may be a unique plesio-                       fashion) may be much longer than the time indicated for
morphic trait in bivalves but reversals in the route of                       the Mytilidae. Given that the taxonomic split between
transmission    of gender-associated       types may occur pe-                the Pyganodon and Fusconaia lineages occurred at least
riodically. These hypothetical       transitions were referred                 100 MYA, Hoeh et al. (1996) deduced that the M and
to as “masculinization”       or “feminization”         events by             F mitotypes in these species are at least 100 Myr old.
Hoeh et al. (1996). The strongest evidence in support of                      In contrast, Rawson and Hilbish (1995) estimated the
masculinization     events comes from the phylogenetic           af-          time of the split between the most common M and F
finity of the M. trossulus     type M-O and the M. e&&s                       mitotypes in the M. edulis species complex at 5.3 MYA.
type M-A with their respective conspecific F types (fig.                      Hoeh et al. (1996) estimated that the formation of the
2). The derived position of these paternally transmitted                      M and F mitotypes in Geukensia was of a similar age.
mitochondrial     genomes within M. edulis and M. tros-                       The newly masculinized         M mitotypes in Mytilus are
sulus relative    to the maternally      transmitted     mitotypes            clearly of even more recent origin. In contrast to the
coupled with the close genetic similarity between these                       large divergences     between the standard conspecific M
conspecific M and F types suggests that the paternally                        and F mitotypes in M. edulis and A4. trossulus (ca. 25%-
inherited mitotypes were recently spawned from female                         30%), the newly masculinized       mitotypes have diverged
mitotypes (fig. 2 and table 1). Similarly, the phyloge-                       from their conspecific female types by less than 2.8%
netic position of the M. trossulus and M. edulis M types                      (table 1).
relative to the M. californianus F type (fig. 3) also in-                            The sequence of events that led to masculinization
dicates another instance of masculinization             of female             remains unknown. However, one possible mechanism is
mtDNA, assuming that this is the true phylogeny                   of          supported by recent studies of mtDNA transmission           in
these genomes (see below). By extension, we propose                           Mytilus (Zouros et al. 1994b; Saavedra, Reynero, and
that the same process could have occurred repeatedly in                       Zouros 1997). In these crosses, some males failed to
the history of the bivalves. This hypothesis is summa-                        pass either the M or F mitotype to their sons. A small
rized in figure 4, which is a synthesis of phylogenetic                       percentage of sons from other males also failed to inherit
information from this study and Hoeh et al. (1996) com-                       mtDNA from their fathers. The overall rate of M-neg-
bined with the most parsimonious           reconstruction    of an-           ative males in these crosses was about 20%. Although
cestral gender states. Transitions        4, 5, and 6 are only                the information existing so far is limited to a few cases,
                                                                                                     Masculinization   of mtDNA     in Mytilus    965

                                                                         M. calijomianus               M. edulis             M. trossulus    Taxon
M-negative        males produce        sperm that contains         the
mtDNA of the mother (Saavedra, Reynero, and Zouros                            M     F                   M   F M-A            M M-O F
 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 re-
versals 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 mi-
totype (thereby eliminating          one masculinization       event),
the length of the tree increases considerably             (from 689
to 710 steps). Furthermore,         to accept an alternate topol-
ogy 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 spe-
cies boundaries       to account for the pattern of mitotype
relationships.     Based on the results of a survey of 201                      FIG. 5.-Hypothetical     reconstruction of speciation, mitotype mas-
“pure” and “hybrid” M. edulis and M. trossulus mus-                      culinization,   and mitotype extinction events which could explain the
sels from the wild, Saavedra et al. (1996) concluded that                observed phylogenetic relationships of the M and F mitotypes sampled
                                                                         in extant members of the genus A4yriZus.The heavy solid lines outline
there are intrinsic barriers to the exchange of mtDNA
                                                                         organismal phylogeny. The light solid lines represent F mitotype phy-
between these two species. The bias in favor of mas-                     logeny while the dashed lines represent M mitotype phylogeny. Chro-
culinization     as opposed to feminization          events is also      nology: A, An ancestral Mytilus lineage displays DUI. B, Allopatric
consistent with the data currently available concerning                  speciation occurs such that an ancestral species bifurcates to produce
within-individual       mitotype distributions     in Mytilus. The       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
number of M-positive females is much smaller than that                   to the M. edulislhf trossulus complex and before the separation of h4.
of M-negative males, and the amount of M mitotype in                     edulis and M. trossulus, the F mitotype lineage spawned an M mitotype
the former is very small, whereas in the latter the F                    via a masculinization     event. D, The common ancestor of 44. edulis and
molecule is 100%. Furthermore,             there is, as yet, no ev-      M. trossulus would then have passed through a transitional state of
                                                                         polymorphism      for the newly masculinized      M mitotype and the pre-
idence of eggs containing           M molecules but, as noted,
                                                                         existing M mitotype. E, The original M mitotype in the M. edulis/M.
there is evidence that M-negative males produce sperm                    trossulus complex went on to extinction. F, Allopatric speciation lead-
that contains the F molecule (Saavedra, Reynero, and                     ing to M. edulis and M. trossulus. G, Masculinization        event giving rise
Zouros 1997). While alternate interpretations               invoking     to the M. edufis M-A mitotype. H, Masculinization           event giving rise
feminization      events do not appear to be in accord with              to the M. trossulus M-O mitotype. The M. trossulus F-O mitotype has
                                                                         been omitted to simplify the presentation.
our limited data from the wild or from laboratory cross-
es, they cannot, for the moment, be completely discount-
ed.                                                                      assume a standard mode of allopatric         speciation  in
       Role reversal of an F mitotype to yield a newly                   which an ancestral DUI-containing     species (fig. 5A) bi-
masculinized       M mitotype is presumably the first step in            furcates to produce two descendant lineages (fig. 5B).
a process which resets, initially to zero, the amount of                 One of these leads to the M. eduZis/M. trossulus com-
sequence divergence          (and apparent time since diver-             plex, and the other leads to M. californianus.     Presum-
gence) between a species’ M and F mitotypes. The sec-                    ably, each of these descendant    lineages contained ho-
ond step is a transient state of polymorphism               in which     mologous (orthologous) M and F mitotypes. In the line
both new and old M mitotypes coexist in the population.                  leading to the M. eduZislA4. trossulus complex and be-
The third step is replacement          of the old M mitotype by          fore the separation of M. edulis and M. trossulus, the F
a newly masculinized          type (see Hoeh et al. 1996, fig.           lineage spawned an M mitotype via a masculinization
3). We can illustrate how these stages would have pro-                   event (fig. 5c). The common ancestor of M. edulis and
ceeded for the species of the genus Mytilus (fig. 5). First,             M. trossulus would then have passed through a transi-
966   Hoeh et al.


 tional state of polymorphism   for the newly masculinized       GELLER, J. B. 1994. Sex-specific mitochondrial       DNA haplo-
M mitotype and the preexisting        M mitotype (fig. 5D).         types and heteroplasmy      in Mytilus trossulus and Mytilus
Finally, the original M mitotype presumably went on to              galloprovincialis  populations. Mol. Mar. Biol. Biotechnol.
extinction (fig. 5E), since we have not, as yet, found an           3:334-337.
                                                                 HILLIS, D. M., and J. J. BULL. 1993. An empirical test of boot-
M mitotype in the M. eduZislA4. trossulus complex that
                                                                     strapping as a method for assessing confidence in phylo-
is a sister mitotype to the A4. californianus M mitotype.
                                                                    genetic analysis. Syst. Biol. 42: 182-192.
As mentioned      above, a consequence    of this process is     HILLIS, D. M., J. I? HUELSENBECK, and C. W. CUNNINGHAM.
that the apparent origin of DUI (i.e., divergence of F               1994. Application and accuracy of molecular phylogenies.
and M mitotypes) in the A4. eduZis/M. trossulus species             Science 264:67 l-677.
complex is moved forward in time to the point of the             HOEH, W. R., D. T STEWART, B. W. SUTHERLAND,and E. Zou-
most recent masculinization     event.                              ROS. 1996. Multiple origins of gender-associated    mitochon-
       From a population genetics point of view, we want            drial DNA lineages in bivalves (Mollusca: Bivalvia). Evo-
to know if the replacement      of the old M lineage by a           lution 50:2276-2286.
newly masculinized      lineage is a stochastic event, or if     HUELSENBECK, J. I?, and D. M. HILLIS. 1993. Success of phy-
                                                                    logenetic methods in the four-taxon case. Syst. Biol. 42:
the latter has intrinsic advantages over the former. It is
                                                                    247-264.
well established that the M lineage evolves faster than
                                                                 HURST, L. D., and R. E HOEKSTRA. 1994. Shellfish genes kept
the F lineage, and we have presented evidence that the              in line. Nature 368:811-812.
reason for this is relaxed selection against the M type          KUMAR, K., K. TAMURA, and M. NEI. 1993. MEGA: molecular
(Stewart et al. 1996). This mechanism might favor the               evolutionary genetics analysis. Version 1.02. Pennsylvania
accumulation     of slightly deleterious   mutations    which       State University, University Park.
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competition with a new M type. On the other hand, this              chondrial DNA differentiation      far exceeds maternal DNA
could lead to very frequent masculinization      events. This       and allozyme differentiation    in the freshwater mussel, An-
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                                                                 MADDISON, W. I?, M. J. DONOGHUE, and D. R. MADDISON.
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                                                                     1984. Outgroup analysis and parsimony. Syst. Zool. 33:
newly spawned molecules quickly replace the old mol-                83-103.
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gence of M and F molecules. In such systems, the ex-                ysis of phylogeny and character evolution. Version 3.01.
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                                                                 PALUMBI, S. R., A. l? MARTIN, S. ROMANO, W. 0. MCMILLAN,
Acknowledgments                                                     L. STICE, and G. GRABOWSKI. 1991. The simple fool’ guide
                                                                                                                           s
                                                                    to PCR. Department of Zoology, University of Hawaii, Ho-
      We thank the following for assistance in the pro-             nolulu.
curement and/or maintenance      of specimens: W. Borge-         QUESADA, H., D. A. G. SKIBINSKI, and D. 0. E SKIBINSKI.
son, D. Cook, D. Hedgecock,         C. Herbinger, E. Ken-            1996. Sex-biased heteroplasmy and mitochondrial DNA in
chington, J. Maunder, R. Noseworthy, and R. Trdan. A.               the mussel Myths       galloprovincialis   Lmk. Curt-. Genet.
Ball, S. Baldauf, D. Cook, and M. Dillon kindly pro-                29~423-426.
vided assistance   with laboratory    procedures  and (or)       RAWSON, I? D., and T. J. HILBISH. 1995. Evolutionary relation-
provided access to the facilities of the Marine Gene                ships among the male and female mitochondrial DNA lin-
Probe Laboratory, Dalhousie University. We also thank               eages in the Myths        edulis species complex.      Mol. Biol.
                                                                    Evol. 12:893-901.
Andrew Martin and an anonymous reviewer for numer-
                                                                 SAAVEDRA, C., M. REYNERO, and E. ZOUROS. 1997. Male-de-
ous constructive  comments on this manuscript. This re-
                                                                    pendent doubly uniparental inheritance of mitochondrial
search was supported by grants from the Natural Sci-                DNA and female-dependent        sex-ratio in the mussel Myths
ences and Engineering      Research Council of Canada               galloprovincialis.  Genetics 145: 1073-1082.
(NSERC) to E.Z. and by the Research Development                  SAAVEDRA, C., D. T. STEWART, R. R. STANWOOD,and E. Zou-
Fund (Dalhousie     University)    to W.R.H. and D.T.S.             ROS. 1996. Species-specific     segregation of gender-associat-
W.R.H. and D.T.S. were supported by postdoctoral fel-               ed mitochondrial DNA types in an area where two mussel
lowships from NSERC. C.S. was supported by postdoc-                 species (Myths      edulis and Myths        trossulus) hybridize.
toral fellowships  from the Conselleria      de Education,          Genetics 143: 1359-l 367.
Xunta de Galicia (Spain) and the Ministerio de Educa-            SKIBINSKI, D. 0. E, C. GALLAGHER, and C. M. BEYNON.
                                                                    1994~. Mitochondrial      DNA inheritance. Nature 368: 8 17-
cion y Ciencia (Spain).
                                                                    818.
                                                                 -.        1994b. Sex-limited mitochondrial DNA transmission
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