Molecular Phylogeny of Philippin

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					                                                            Molecular Phylogeny of Philippine Freshwater
                                                            Sardines Based on Mitochondrial DNA
                                                            Analysis
                                                            I. E. Samonte, R. C. Pagulayan, and W. E. Mayer




                                                            The commercially important Sardinella species (family Clupeidae or herrings) usu-
                                                            ally thrive in marine environments. An exception is Sardinella tawilis of Taal Lake,
                                                            Batangas, Philippines, the only known freshwater sardine. This species is believed
                                                            to have immigrated from Balayan Bay to the lake when it was formed in the course
                                                            of volcanic eruptions some 240 years ago. To determine the relationship of S. taw-
                                                            ilis to the marine species S. albella, S. fimbriata, and S. longiceps from the Balayan
                                                            Bay we sequenced 358 bp of the cytochrome b gene and the mitochondrial control
                                                            region. The cytochrome b gene was highly conserved and contained little phylo-
                                                            genetic information. The control region sequences, however, demonstrated two
                                                            highly diversified main haplotypes grouping S. tawilis with S. albella, as shown by
                                                            maximum parsimony and neighbor-joining analysis. The haplotypes are character-
                                                            ized by the presence of an 81 bp indel and up to eight 35 bp tandem repeat ele-
                                                            ments. The repeat copy number varied within individuals of S. tawilis and S. albella,
                                                            thus showing heteroplasmy in these two species only. The analysis of two sub-
                                                            populations of S. tawilis revealed restricted substitutions that may indicate the be-
                                                            ginning of genetic differentiation of the two subpopulations.

                                                            Members of the family Clupeidae, or her-       by the collapse of a large volcanic crater
                                                            rings (order Clupeiformes, subclass Actin-     and by subsidence, and whose morphom-
                                                            opterygii), are fish with a compressed          etry has been modified by subsequent vol-
                                                            streamlined body, a single soft-rayed dor-     canic activities, especially by a powerful
                                                            sal fin, and protruding scales. They are        eruption in 1754 ( Hargrove 1991). The 10th
                                                            found worldwide in temperate and tropi-        century lake, connected to Balayan Bay
                                                            cal waters where they mainly move in           through a wide channel, has now rear-
                                                            large schools feeding on plankton. Most of     ranged its shape and narrowed its outlet to
                                                            the species live in the marine environ-        form the Pansipit River, the sole outlet from
                                                            ment, but some, like the American gizzard      its southwest corner ( Kira 1995). Like oth-
                                                            shad (Dorosama cepedianum), occur only         er members of the genus, tawilis has an ob-
                                                            in freshwater systems. The family Clupei-      long to nearly elongated body, equal jaws,
                                                            dae includes the subfamilies Alosinae or       large mouth, lacks lateral lines, and has
                                                            shads, the Pellonulinae or freshwater her-     thin deciduous scales (Munro 1955). Be-
                                                            rings, and the Clupeinae with the genera       cause of its small size (10–20 cm) it does
                                                            Clupea, Sardina, and Sardinops.                not command much interest economically,
                                                               Little is known about the phylogenetic      but its year-round presence makes it im-
                                                            relationships among the commercially im-       portant to fishers who depend solely on
                                                            portant Sardinella species ( Family Clupei-    the lake for their daily subsistence. Interest
                                                            dei, Suborder Clupeoidei), especially at the   in this species has arisen mainly because
                              ¨
From the Max-Planck-Institut fur Biologie, Abteilung Im-    molecular level. In the Philippines, eight     of a recent report of human intervention in
                                           ¨
mungenetik, Corrensstrasse 42, D-72076 Tubingen, Ger-       members of this genus were identified and       the lake which threatens its fishery re-
many (Samonte and Mayer), and the Institute of Biol-
ogy, University of the Philippines–Diliman, Diliman,
                                                            collectively called sardines (Conlu 1986).     sources ( Villanueva et al. 1996). Also the
Quezon City, Philippines (Pagulayan). We thank Dr. Jan      All of these species are thriving in the ma-   recent reclassification of the species from
Klein for his support and stimulating discussions. I.E.S.   rine environment except Sardinella tawilis     Harengula to Sardinella on the basis of mor-
was supported by a short-term scholarship from the
Deutscher Akademischer Austauschdienst ( DAAD) and          (Whitehead 1985), the only known fresh-        phoanatomic features makes it an interest-
the Engineering and Science Education Program ( ESEP)       water sardine, endemic to Taal Lake, Batan-    ing subject of a molecular study ( FishBase
of the Department of Science and Technology ( DOST ),       gas, Philippines. Known locally as tawilis,    1995).
Philippines. Address correspondence to Werner E.
Mayer at the address above or e-mail: werner.mayer@         this species is believed to have immigrated       Analyses of the mitochondrial DNA
tuebingen.mpg.de.                                           from Balayan Bay to Taal Lake. The said        (mtDNA) sequences have proved to be a
  2000 The American Genetic Association 91:247–253          lake was a caldera lake that formed partly     valuable tool in addressing the problems



                                                                                                                                                     247
    Table 1. Primers used for the amplification of the cytochrome b gene and the control region of the
    sardines

    Primer designation               Sequence

    Cytochrome b:
     cytbLa                          5 -CCCCTCAGAATGATATTTGTCCTCA-3
     cytbHa                          5 -CCATCCAACATCTCAGCATGATGAAA-3
    Control region:
     L15926a                         5   -TTACACCAGTCTTGTAAACC-3
     H16498b                         5   -CCTGAAGTAGGAACCAGATG-3
     Q                               5   -GGGCGGATCCCACCACTAGCTCCCAAA-3
     B                               5   -ACGCTGGAAAGAACGCCCGGCATGG-3
     J                               5   -TTTGGTTCCTATTTCAGGGCCA-3
     N                               5   -GGGGCGCGGATCCCATCCTAATATCTTCAG-3
     A88c                            5   -GCAAAACTCTCACCAACTCATC-3
     A87c                            5   -GGGGTTTCATGGTGAACT-3

a
  Primer designed by Kocher et al. (1989) except that the restriction site was deleted.
b
  Primer designed by Meyer and Wilson (1990).
c
  Internal primers designed from the generated sequences. The remaining primers are those used by Lee at al.
  (1995).
                                                                                                               Figure 1. Geography of Taal Lake showing the Pan-
                                                                                                               sipit River and portions of Balayan Bay and Batangas
                                                                                                               Bay. The location of the Talisay and Agoncillo parts is
    that S. tawilis faces. Features of this mol-         1995). The variable portions of the control           indicated.
    ecule, such as its compact organization              region contain varying numbers of tandem
    (Russell 1994), its elevated rate of muta-           repeat sequences ( Broughton and Dowl-
                                                                                                               tions of S. tawilis were obtained from the
    tion ( Brown 1983), its primarily maternal           ing 1994; Lee et al. 1995) which are the
                                                                                                               Talisay and Agoncillo portions of Taal
    inheritance, and the absence of recombi-             targets of frequent mutations, particularly
                                                                                                               Lake ( Figure 1). S. albella and S. fimbriata
    nation (Moritz et al. 1987), make it a valu-         deletions, insertions, and duplications
                                                                                                               from Balayan Bay were procured from fish
    able tool in evolutionary and population             ( Brown 1983; Harrison 1989). Its fast evo-
                                                                                                               vendors in Lemery, Batangas. S. longiceps,
    studies.                                             lutionary rate makes the control region a
                                                                                                               caught in Cebu Bay, was obtained from the
       Fish mtDNA ranges in length from 15.2             suitable marker for resolving relatively re-
                                                                                                               Malabon Fish Port in Malabon, Metro Ma-
    to 19.8 kb ( Billington and Hebert 1991)             cent divergences of closely related species
                                                                                                               nila. Muscle and gonads were dissected
    and contains information specifying the              and populations ( Zischler et al. 1995).
                                                                                                               out and preserved in 85% ethanol. These
    two rRNAs (16s and 12S), 22 tRNAs, 13                Analyses of mitochondrial genes have
                                                                                                               were then brought to the Max-Planck In-
    polypeptides, as well as the dissociation            been facilitated by the discovery of the
                                                                                                               stitute for Biology for analysis.
    ( D) loop, also referred to as the control           polymerase chain reaction (PCR), which
    region. The proteins it encodes include              enables specific sections of the genes to
                                                                                                               DNA Extraction
    NADH dehydrogenase, cytochrome oxi-                  be routinely investigated (Carr and Mar-
                                                                                                               Total DNA of the ethanol-preserved sam-
    dase, cytochrome b (cytb), and ATPase 6              shall 1991; Kocher et al. 1989) from minute
                                                                                                               ples was extracted according to the
    and 8 (Gyllensten and Wilson 1987). Of               quantities of tissue samples (McVeigh et
                                                                                                               NucleoSpin C T protocol (Macherey-Na-
    these, cytochrome b ( Barlett and David-             al. 1991) to yield high resolution informa-
                                                                                                                     ¨
                                                                                                               gel, Duren, Germany). MtDNA was extract-
    son 1991) together with the control region           tion about any variation that may exist in
                                                                                                               ed using the modified phenol extraction
    ( Zhu et al. 1994) have become widely                the DNA ( Hartley et al. 1992).
                                                                                                               procedures of Gonzales Villasenor et al.
    used molecular markers. In vertebrates,                 The aim of this study was to determine
                                                                                                               (1990), Chapman and Powers (1984), and
    the rate of the cytb gene evolution is esti-         the phylogenetic relationship of S. tawilis
                                                                                                               Bernatchez et al. (1988). The extracted
    mated to be between 1% and 2.5% substi-              to the marine sardines S. albella (White-
                                                                                                               mtDNA was precipitated with ethanol and
    tutions per one million year ( Irwin et al.          head and Wongratana 1986), S. fimbriata
                                                                                                               resuspended in 50 l 10 mM TE buffer (pH
    1991; Martin et al. 1992) which means that           (Whitehead 1985), and S. longiceps (White-
                                                                                                               8.5). Yields were typically 800 g DNA/ml
    approximately one hundred thousand                   head 1985), and the genetic variability be-
                                                                                                               of TE.
    years are needed before one might confi-              tween populations of S. tawilis from the
    dently expect a substitution to differenti-          mtDNA sequences. The latter information
                                                                                                               PCR Amplification
    ate closely related mtDNA types (Palumbi             was analyzed for fish management purpos-
                                                                                                               The primer sets used to PCR amplify (Saiki
    and Kessing 1991). This relatively slow              es.
                                                                                                               et al. 1988) a segment of the mitochondrial
    evolution of the cytb gene makes it a suit-
                                                                                                               cytochrome b gene (cytb) and the mito-
    able marker for resolving deeper phylo-
                                                         Materials and Methods
    genetic relationships among different taxa
    (Meyer and Wilson 1990). Unlike cytb, the            Collection of Fish Samples
    mitochondrial control region does not                Four Philippine sardine species, S. tawilis,
    code for proteins (Avise et al. 1987) and            S. albella, S. fimbriata, and S. longiceps,
    has only short sequence blocks conserved             were collected and transported to the Nat-
    among distant taxa ( Brown et al. 1993;              ural Sciences Research Institute’s labora-
    Clayton 1982; Lee et al. 1995; Saccone et            tory for identification and dissection. Rau
                                                                                                               Figure 2. Schematic representation of the mitochon-
    al. 1987), presumably because of relaxed             and Rau (1980) and Conlu (1986) served                drial control region. Primers used for PCR and se-
    functional constraints (Alves-Gomes et al.           as bases for identification. Two popula-               quencing are indicated by arrows.




    248 The Journal of Heredity 2000:91(3)
chondrial control region are shown in Ta-      ware (version 0.6, written by Don Gilbert,       populations of S. tawilis, as shown in Fig-
ble 1 and in Figure 2. Hot-Start PCR was       Indiana University, Bloomington, IN) and         ure 2.
performed in a 50 l reaction containing        the alignments were then corrected man-             The complete control region sequence
Mg2 -free PCR buffer [60 mM Tris, 15 mM        ually. For the control region analyses, Alo-     of the Philippine sardines is 804 nucleo-
( NH4)2SO4, pH 8.5 ( Invitrogen, Leek, The     sa sapidissima, Alosa pseudoharengus, and        tides long for S. fimbriata and S. longiceps,
Netherlands)], 250 M dNTPs, 0.5 M of           Clupea harengus as the most closely relat-       while it ranges from 1231 to 1418 nucleo-
each primer, 1.0 l DNA template, and 2.5       ed sequences in the GenBank database             tides in S. albella and the two populations
units Taq polymerase (Pharmacia Bio-           served as outgroups. Maximum parsimony           of S. tawilis due to the presence of an 81
tech). Magnesium was supplied to the re-       (MP) analysis was performed using the            bp insertion and several copies of a 35 bp
actions by adding HotWax-Mg2 beads ( In-       branch and bound search algorithm of the         repetitive element near the tRNAPro region
vitrogen). Amplification was performed in       PAUP 3.1.1 software (Swofford 1993). The         ( Figure 3). At its 5 end the 81 bp insertion
the PTC-200 thermal cycler (MJ Research,       topology of the consensus tree was eval-         repeats the 32 bp preceding the insertion
Watertown, MA, and Biozym, Hess. Old-          uated by 500 bootstrap replications.             site. The copy number of the 35 bp repet-
endorf, Germany). The amplification pro-        Neighbor-joining ( NJ) trees (Saitou and         itive element varied from five to eight in
file consisted of a 3-min preliminary de-       Nei 1987) were constructed from a Kimura         the sequenced fragments. This variation
naturation at 94 C, followed by 30 cycles      two-parameter distance matrix ( Kimura           was also observed within individuals,
of denaturation at 94 C for 30 s, primer an-   1980) using the MEGA program ( Kumar et          thereby producing more than one form of
nealing at 48 C–55 C for 45 s, and primer      al. 1993). Input orders of the taxa were var-    mtDNA per fish. To verify this phenome-
extension at 72 C for 2 min. An extension      ied to test the robustness of the trees. The     non of heteroplasmy, the PCR products
step at 72 C for 5 min completed the re-       reliability of the tree topology was as-         from tissue samples of all species and
action. After thermal cycling, the PCR         sessed by 500 bootstrap replications ( Fel-      from cloned control region fragments were
products were separated on 1% agarose          senstein 1985). The number of transitions        electrophoresed through a 2.5% NuSieve 3:
gel ( NEEO ultra-quality, Roth, Karlsruhe,     and transversions was calculated using           1 agarose gel with high resolving power.
Germany) and retrieved using the QIA-          the MEGA program.                                The analysis revealed three to five bands
quick Gel Extraction Kit (QIAGEN, Hilden,                                                       ranging from 800 to 1100 bp in mtDNA
Germany).                                                                                       samples from S. albella and S. tawilis, but
                                               Results                                          only single bands in S. longiceps or S. fim-
Cloning                                                                                         briata and in the samples of cloned control
The purified PCR products were ligated          Nucleotide Variation in the Cytochrome           region fragments from either species, thus
into pGEM-T Easy Vector (Promega, Mann-        b Gene                                           confirming the presence of heteroplasmic
heim, Germany) and transformed into E.         The amplification of a 359-nucleotide long        mtDNA in S. albella and S. tawilis and ex-
coli JM109 according to manufacturer’s in-     cytb fragment from fishes of two popula-          cluding PCR artifacts.
structions. Plasmid DNA from recombi-          tions of the freshwater sardine species S.          Comparison of the sardine control re-
nant clones was isolated using the QIA-        tawilis from Taal Lake and three marine          gion sequences in Figure 3 shows low
GEN Plasmid Mini Kit.                          sardine species from the Balayan Bay             similarity between the two main haplo-
                                               produced identical sequences except for          typic sequences. The highest variability
Sequencing                                     single individual-specific substitutions          resides in the first 940 nucleotides fol-
Single-stranded plasmid DNA was se-            (GenBank accession codes AF1014944–              lowed by low variability in the rest of the
quenced using ALF DNA sequencer (Phar-         AF104948). Only a single transition at po-       sequence. Conserved sequence blocks
macia Biotech) and/or LI-COR Automated         sition 166 was shared by two individuals         (CSBs), CSB-D and CSB-2, homologous to
Sequencer 4200 (MWG-Biotech, Ebers-            from the northern population of S. tawilis       those of other vertebrates ( Lee et al.
berg, Germany). For ALF, sequencing re-        ( Talisay). It occurred at a nonsynony-          1995), are present together with the
actions were prepared using either the         mous site leading to the conservative            GTGGG-box.
AutoRead 200 Sequencing Kit (Pharma-           amino acid replacement Ile to Val.
cia Biotech) or the SequiTherm EXCEL                                                            Sequence-Based Phylogeny
Long-Read Premix DNA Sequencing Kit-           Nucleotide Variation in the Control              Control region. Only mitochondrial control
ALF ( Epicentre Technologies, Biozym). LI-     Region                                           sequences were informative for phyloge-
COR sequencing reactions were prepared         For the amplification of the mitochondrial        netic analysis. The control region analysis
using the SequiTherm Long-Read DNA             control region of the different sardine spe-     was divided into two parts. First, the seg-
Sequencing Kit-LC ( Epicentre Technolo-        cies, distinct primer sets had to be ap-         ment between the tRNAPro gene and the
gies, Biozym) with primers labeled with        plied, suggesting haplotypic variation be-       conserved sequence blocks was used for
two different fluorescent dyes ( IRD 700        tween species. The primer pair L15926            the assessment of the phylogenetic posi-
and IRD 800, MWG-Biotech).                     and H16498 amplified the first two-thirds          tion of the sardines because of the large
                                               of the control region of S. fimbriata and S.      number of informative sites and because
Phylogenetic Analysis/Sequence                 longiceps to produce a fragment of 600 bp,       corresponding sequences from related
Analysis                                       whereas the primer combination Q and B           fish species were available in the GenBank
Only sequences obtained by at least two        generated the corresponding product in S.        sequence database. Second, all homolo-
independent PCRs were considered. The          albella and the two populations of S. taw-       gous segments of the control region were
cytb and the control region sequences          ilis. The remaining portions were amplified       used for a more detailed analysis within
were aligned together with related se-         with the primer pairs A87/A88 for the two        the genus Sardinella. In the first part, the
quences available from the GenBank da-         populations of S. tawilis, J/A88 for all the     analysis was restricted to the alignable
tabase with the help of the SeqPup soft-       sardines, and J/N for S. albella and the two     segments (positions 699 to 961 in Figure



                                                                                          Samonte et al • Philippine Freshwater Sardine Phylogeny 249
Figure 3. Sequence alignment of the sardine mitochondrial control region. Representative sardine sequences were aligned to control region sequences of A. sapidissima, A.
pseudoharengus, and C. harengus. The sardine sequences are distinguished by a number for the individual specimen, separated by a dot from the sequence number. The two
subpopulations of S. tawilis are indicated by a T ( Talisay) and an A (Agoncillo). A dash (–) indicates identity to the sequence on top, an asterisk (*) an indel, a dot (.) missing
information, a ( N) undetermined nucleotide at the primer annealing site. The 32 bp segment preceding the 81 bp insertion site is underlined, solid bars indicate the 35 bp
repeat units, the shaded bar the central conserved region. Conserved sequence blocks are boxed. The sequences reported in this article have been deposited in the GenBank
database (accession codes AF104949–AF104969).




3) of the two main sardine haplotypes and                    cluster together in one branch, and the                       while others seem to represent intraspe-
the sequences of the most closely related                    freshwater species S. tawilis and the ma-                     cific variants.
Clupeidae ( herrings), Alosa sapidissima                     rine species S. albella group in a separate
(GenBank accession U12061), A. pseudo-                       branch. The clades are supported by high
                                                                                                                           Discussion
harengus (accession U12067), and Clupea                      bootstrap values.
harengus (accession U12062), excluding all                      To evaluate the relationships within the                   Structural Analysis
repeat elements. The alignment of the re-                    genus Sardinella, the nonrepeat portions                      The amplified segments of the Sardinella
maining 263 nucleotides was used to re-                      of the control region of S. tawilis and S.                    mtDNA showed features characteristic of
construct phylogenetic trees by the neigh-                   albella were aligned (positions 45–160,                       the respective regions. The selective pres-
bor-joining and maximum parsimony                            241–487, 594–1058) . A total number of 30                     sure exerted on the protein-coding cytb
methods ( Figure 4). Both trees show es-                     variable nucleotide sites was found in the                    gene kept its sequence conserved among
sentially the same topology and reveal the                   626 nucleotides considered, some of                           the sardines. In stark contrast, in the con-
divergence of the sardine control region                     which distinguish S. albella from S. tawilis                  trol region, even primer sets known to am-
into two major haplotypes evident already                    (substitutions at positions 100, 263, 279)                    plify mtDNA from species as distantly re-
from the presence or absence of the re-                      or the two subpopulations of S. tawilis                       lated as teleost fishes and primates failed
peat elements: S. fimbriata and S. longiceps                  from each other (positions 778, 846),                         to amplify two of the sardine species. The



250 The Journal of Heredity 2000:91(3)
                                                                                             Figure 4. (A) Maximum parsimony tree. The tree was
                                                                                             constructed from alignable segments of clupeid control
                                                                                             regions using the branch-and-bound option of the PAUP
                                                                                             program. Numbers at the nodes indicate the percent
                                                                                             recovery in 500 bootstrap replications.


                                                                                             due to multiple copies of repeat units. The
                                                                                             first of these repeat units, an 81 bp inser-
                                                                                             tion, was found in some S. tawilis sequenc-
                                                                                             es and in S. albella. The second repeat el-
                                                                                             ement, a tandem repeat of 35 bp, was
                                                                                             present in up to eight copies in S. tawilis
                                                                                             and S. albella. The single repeat units
                                                                                             showed low or no sequence variation from
                                                                                             each other indicating a recent divergence,
                                                                                             whereas the similarity to the monomeric
                                                                                             element in S. fimbriata and S. longiceps
                                                                                             was rather low. To calculate the ratio of
                                                                                             transitions to transversions in the control
                                                                                             region, 626 repeat-free nucleotides from S.
                                                                                             tawilis and S. albella were aligned. Six to
                                                                                             17 transitions and zero to five transver-
                                                                                             sions were observed. The mean ratio of
                                                                                             transitions to transversions in the control
                                                                                             region thus was 4.3, suggesting no satu-
                                                                                             ration effects in the Sardinella sequences.

                                                                                             Heteroplasmy
                                                                                             Copy number polymorphism ( heteroplas-
                                                                                             my) of the 35 bp repeat element was found




Figure 3. Continued.



structural organization of the control re-       These segments include the first 13 nu-
gion in S. longiceps and S. fimbriata is dra-   cleotides after the tRNAPro gene and the
matically different from that in S. albella    stretch from the central conserved region     Figure 4. (B) Neighbor-joining tree. A Kimura two-pa-
and S. tawilis, thus defining two different     to the tRNAPhe gene. The variable portion,    rameter distance matrix of the control region sequenc-
major haplotypes, although homologous          which encompasses sequences without           es was used to construct a neighbor-joining tree using
                                                                                             the MEGA program and the pairwise deletion option
segments clearly identify all four species     similarity between the two haplotypes, is     for missing information. Numbers at the nodes indicate
as closely related to each other.              mainly characterized by length variation      the percent recovery in 500 bootstrap replications.




                                                                                       Samonte et al • Philippine Freshwater Sardine Phylogeny 251
to be present within 17 tested individuals      cies cannot be dismissed. Second, the two                  FishBase, 1995. Fishbase: a biological database on fish,
                                                                                                           version 1.2. Manila, Philippines: ICLARM.
of both populations of S. tawilis and 7 in-     subpopulations of S. tawilis show indica-
                                                                                                           Gonzalez Villasenor LI, Burkhoff AM, Corces V, and
dividuals of S. albella (data not shown).       tions of genetic differentiation. The pres-                Powers DA, 1990. Characterization of cloned mitochon-
Heteroplasmy has been noticed before in         ence of a population-restricted substitu-                  drial DNA from the teleost Fundulus heteroclitus and its
various fish species, among them Atlantic        tion in the control region is a first hint for              usefulness as an interspecies hybridization probe. Can
                                                                                                           J Fish Aquat Sci 43:1866–1872.
      ´
cod (Arnason and Rand 1992), sturgeon           genetic separation, possibly caused by re-
                                                                                                           Gyllensten U and Wilson AC, 1987. Mitochondrial DNA
( Brown et al. 1992; Miracle and Campton        duced fish migration across a shallow bar-                  of salmonids: inter- and intraspecific variability detect-
1995), and redfishes ( Bentzen et al. 1998).     rier in the middle of the lake. A more ex-                 ed with restriction endonucleases. In: Population ge-
The occurrence of heteroplasmic mtDNA           tended investigation with large sample                     netics and fishery management. Seattle: University of
                                                                                                           Washington Press; 302–317.
in only two of the sardine species and the      sizes is needed to answer these questions.
                                                                                                           Hargrove TR, 1991. The mysteries of Taal. Flagstaff, AZ:
virtual identity of the repeat sequences                                                                   AGCOM International.
suggest a recent origin of the tandem re-
                                                                                                           Harrison RG, 1989. Animal mitochondrial DNA as a ge-
peat array.                                     References                                                 netic marker in population and evolutionary biology.
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                                                                                                                   Samonte et al • Philippine Freshwater Sardine Phylogeny 253

				
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