Supplemental data 3.doc.—A brief account of deletions and pseudogenes. WHEN
THE four short (~1.3 kb) PCR amplicons were purified, sequenced and aligned with
full-length SSU standards, they were found to lack a stretch of 500 bp or more,
starting ca. 150 bp from the 5’ end (Supplemental data 2.html). No short SSU
amplicon was ever obtained from Notosaria or Hemithiris, nor from Neorhynchia
D1090. Neorhynchia D1157 appeared to give only a short product, but some
uncertainty arose over its authenticity and it is excluded from the reported alignment
and analyses. Two tested specimens of Basiliola beecheri (D1596 and D1598) and
several individuals of B. lucida all gave only a short fragment, as did the one available
indivdual of Cryptopora gnomon. No comparably short SSU was ever obtained from
a wide range of non-rhynchonellide brachiopods nor were deletions found in any LSU
gene. None of the rhynchonellide SSU rDNA deletions spans an identical region, but
the 5’ ends all occur within a stretch of 26 bp, suggesting that they may depend upon
an undefined common cause operating independently in divergent taxa. With some
DNAs that gave only a short fragment, appropriate variations of PCR conditions
failed to yield a full-length product (details not shown), but this does not imply that
the undeleted sequence is truly absent. The ~1.8 kb fragment from Tethyrhynchia D
1721 was amplified, but was not purified in sufficient quantity for sequencing.
If SSU rDNA molecules bearing such large deletions were to be incorporated into
ribosomes they would be expected to cause a complete loss of function (Buckler et al.,
1997; Weider et al., 2005) But because the ribosome is indispensible for protein
synthesis, and hence for life, it must be assumed that deletion-bearing individuals also
carry genomic copies of undeleted sequence (see below) and that for unknown
reasons it was only in Tethyrhynchia that these were detectably amplified alongside
the deleted copy or copies. Preferential amplification of short PCR fragments can
arise from a variety of causes, of which only one (shortage of Taq polymerase) can
probably be excluded.
In addition to the long deletion, SSU rDNAs from both tested individuals of Basiliola
beecheri (but not B. lucida) also show a 19-nucleotide insertion (apparently a partial
duplication of an adjacent region) about 100 bp away and distal to (3’-ward of) the
long deletion. Either the two tested individuals were closely related (e.g. siblings) or
this feature is a population or species-specific character. Very short (1 or 2
nucleotide), aberrant sequence regions also abut several other deletion termini, and
these details further suggest that the deletion-bearing copies are functionally inactive,
i.e. are pseudogenes. However, the generally enhanced frequency of base substitution
expected in pseudogenes was not evident to inspection (Supplemental_data_2.html) or
in the saturation analysis.
Is the missing sequence nevertheless present?—The essential nature of SSU
rDNA and the regular recovery of both deleted and undeleted copies from
Tethyrhynchia suggested that a search should be made in other taxa for the missing
sequence fragment. A new primer was prepared matching a conserved region roughly
in the middle of the deleted region of Basiliola beecheri (Primer BasF 5’ACTCCTGGCACGGGGAGGT-3’). This forward primer was paired with standard
downstream reverse primers and PCR products with the predicted lengths and fully
normal sequence were obtained (not shown). Thus, at least part of the missing
(deleted) sequence typical of a normal, undeleted version(s) of the SSU gene of this
specimen was shown to be present somewhere in the DNA tested, further suggesting
that the deletion-bearing copies are pseudogenes (Giribet and Wheeler, 2001). No
further investigation was possible.
Mechanism of SSU rDNA deletion and pseudogene formation.—In all cases
where its genomic structure has been explored, the rDNA region comprises tandem
duplications of a regular array of genes in which, owing to homogenizing processes
termed “concerted evolution”, the multiple copies diverge over time much less than
might be expected. The evolutionary origin(s) of the rhynchonellide SSU rDNAs
deletions is undefined, but on present evidence they are of negligible importance,
being functionally complemented by intact sequence elsewhere in the genome,
confined to only part of the extant rhynchonellide lineage, and unknown in the
thousands of metazoans whose SSU rDNA genes have been sequenced via PCR
amplification. Indeed, putative SSU deletions have been described after PCR of
metazoan DNAs only in one clade of myriapods (Giribet and Wheeler, 2001),
although these supposedly deleted regions were later amplified and sequenced
successfully elsewhere (Mallatt et al., 2004, p. 182). We were unable to locate any
reports of metazoan SSU pseudogene sequences amplified directly from genomic
DNA (Web of Science searched May 2012). In a few cases SSU pseudogenes are
reported to have been recovered after preliminary cloning, but these generally lack
confirmation of their genomic origin. Thus, the rhynchonellide SSU rDNA deletions
described here appear to be a quasi-unique metazoan genomic aberration.
The principal mechanism involved in concerted evolution is believed to be intragenomic homologous crossing-over, by which one copy replaces others (Dover, 1982;
Elder and Turner, 1995; Hillis and Dixon, 1991; Weider et al., 2005). Homologous
pairing of tandemly repeated sequence is known to be prone to offset-pairing error
(unequal pairing), such that an exchange may generate a correlated duplication and
deletion. If offset-pairing involves genes in perfect register a change in whole-gene
copy-number occurs, but if it happens between genes in imperfect register an
intragenic duplication and deletion results, and this may be the type of process
underlying the present cases. Because functional rRNA folds into a regular structure
of unpaired loops and paired helices it is conceivable that these structural elements
bear mechanistic relations to the deletion breakpoints, especially in view of the
concentration of 5’ breakpoints into a short region between helices 5 and 6, but the 3’
breakpoints are widely scattered around helix E23-6 (notation for Onchidella celtica
given in Winnepenninckx et al., 1994). The only other potential evidence of deletion
mechanism may be the 19 bp 3’-ward duplicated insertion in Basiliola beecheri and
the small regions of aberrant sequence abutting other deletion ends, but the
duplication may be an independent variation and, taken together, these data do not
provide enough evidence to constrain ideas about deletion mechanism(s).
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