Systematics and evolution of the Acochlidia (Gastropoda, Euthyneura) - a microanatomical approach by means of 3D reconstruction using Amira

Only a small fraction of the estimated species diversity on Earth
already has been discovered, and expected high extinction rates
force biologists to rapid surveys. Molecular barcoding techniques
meet such goals, but taken alone they can hardly connect genetic
discoveries with the large morphology-based body of taxonomic
knowledge that accumulated during centuries. Also, the study of
organismic evolution requires reliable information on phenotypes.
Morphological and biological knowledge on formally described
species can be, however, very heterogeneous regarding both quality
and quantity. Especially problematic are meiofaunal taxa –
biodiversity generally is poorly explored, and species are small,
hard to collect, externally quite uniform and difficult to
distinguish by means of traditional taxonomic techniques. Old
species descriptions often are fragmentary and information may or
may not be reliable. Novel microanatomical imaging techniques
raised the hope to combine the rapid examinations with the
obligatory accuracy and desired comprehensiveness of structural
information obtainable. Among the most successful interstitial
gastropod taxa are the Acochlidia, combining extremely high
morphological and biological diversity with modest species
diversity. The state of research at the beginning of my PhD thesis
considered the Acochlidia as poorly known, enigmatic and
morphologically and biologically aberrant Opisthobranchia,
comprising only 27 valid species. Most of the acochlidian species
are marine mesopsammic and distributed along the coasts of the
world’s oceans, but some species succeeded to invade freshwater
systems on tropical islands. Uniquely among the otherwise
hermaphroditic euthyneurans, some acochlidians have separate sexes.
Previous sampling efforts were biased to European waters and a few
other places that had been visited by experts. Original
descriptions of the acochlidian species were often limited to the
external morphology, the structure of calcareous spicules and the
examination of the radula by light microscopy; furthermore, some
anatomical data were traditionally obtained from
gross-morphological dissection or from paraffin-based histology.
Inner acochlidian classification was controversial and neither
morphology-based nor molecular phylogenetic studies resolved the
origin of this traditional “order” among euthyneuran heterobranch
gastropods. In a case study for Mollusca, and for the first time
for heterobranch gastropods, I comparatively explored the
microanatomy of a representative sampling of known acochlidian
taxonomic diversity applying computer-aided 3D reconstructions with
Amira based on serial semi-thin histological sections. My
dissertation aimed (1) to revise the morphology and taxonomy of
acochlidians, including the most dubiously and incompletely
described species, (2) to generate detailed microanatomical data
sets for comparative purposes, (3) to reconstruct global
acochlidian phylogeny and major traits of their evolution, and (4)
to explore the power and the limits of modern microanatomy against
traditional taxonomy and molecular approaches, and to develop
integrative approaches. Original type material was traced in
museums and institutions according to the literature and loaned for
re-examination whenever possible. Most of the acochlidian species
were re-collected at the type localities. Seven acochlidian species
covering seven of eight families were re-examined in full detail;
other species were studied to the level necessary and possible
considering time constraints; additionally five species were
introduced new to science. The microanatomical part of my
dissertation clearly demonstrates that traditional acochlidian
taxonomy did not provide sufficiently detailed and reliable
anatomical information. In contrast, computer-based 3D
reconstructions with the software Amira are an efficient, powerful
tool for microanatomy, providing a wealth of new data on all major
organ systems of the Acochlidia. Transforming specimens into serial
histological sections is “invasive”, but generates vouchers that
carry testable information. Semithin-sectioning (1-2 µm) and
staining as applied herein provide resolution adequate to trace
relevant organs, ducts and tissues; limits of this method refer to
quantitative detection of fine nerves. The process of preparing
complete 3D models is time consuming, but greatly supports
accurateness of finding and identifying structures and includes
several steps of internal quality control. 3D models, especially
when interactive, are attractive and instructive, comprise
verifiable high-quality data, and revealed considerable amounts of
erroneous data within original species descriptions. Former
outliers – i.e. apparently aberrant and enigmatic species - fit
well into the pattern of known acochlidian species after the
correction of the original data. 3D modeling from serial sections
as applied herein is discussed as the best currently available
method for exploring complex soft part microanatomy in small
invertebrate specimens. Using the verified and supplemented
morphological data, more than 100 morphological characters were
defined and coded for all 27 acochlidian species considered valid
at that time, and 11 euthyneuran outgroups. A cladistic analysis
with PAUP recovered monophyletic Acochlidia originating from an
unresolved basal opisthobranch level. The Acochlidia split into the
Hedylopsacea (Tantulum (Hedylopsis (Pseudunela (Strubellia
(‘Acochlidium’, ‘Palliohedyle’))))) and Microhedylacea (Asperspina
(Pontohedyle, ‘Parhedyle’, ‘Microhedyle’, (Ganitus, Paraganitus))).
The formerly enigmatic Ganitidae, resembling sacoglossan
opisthobranchs by having dagger-like rhachidian radular teeth, were
recovered as highly derived microhedylids. This topology is largely
well-supported, robust to modifications of outgroup taxon sampling,
and in principles was supported by a recent multi-locus molecular
analysis. In addition, molecular analyses revealed the formerly
enigmatic, amphibious Aitengidae also clustering within
hedylopsacean Acochlidia. Although my phylogenetic hypothesis is
not considered definitive, the paraphyly of some of the
traditionally recognized family level taxa induced a preliminary
reclassification of the Acochlidia. Rarely among invertebrates,
morphology-based and molecular acochlidian topologies are
compatible, and thus may closely reflect natural relationships.
Major traits of the acochlidian evolutionary history were
reconstructed tracing character state changes on the tree. The
previous hypothesis of a general regression of morphological
complexity in the Acochlidia applies only for microhedylacean
species. Within Microhedylacea, we confirmed a tendency towards
successive reductions, particularly in the reproductive system.
Species are aphallic, sperm transfer occurs by spermatophores and
dermal fertilisation and the secondary gonochorism evolved once in
the ancestor of the Microhedylidae. In contrast, already basal
hedylopsacean species show a complex excretory system adapted to a
freshwater influenced environment. An evolutionary trait from a
simple unarmed copulatory system towards complex hypodermal
injection systems was recognised culminating in a large, trap-like
spiny rapto-penis of several limnic Acochlidiidae. In spite of a
high level of convergence involved, precise microanatomical data
sets on a vast (yet incomplete) ingroup sampling thus allowed
reconstructing a novel, plausible and detailed hypothesis on
acochlidian phylogeny and evolution. This approach may have
considerable potential also in other groups with similarly small
and rare members that are elusive to molecular studies. Limits of
morphology-based phylogeny concern any subgroups with just limited
information available, old and possibly rapid diversifications,
such as the origin of Acochlidia among Euthyneura, and relatively
recent subgroups with little phenotypical differences fixed. We
show that traditional taxonomy fails to differentiate some
genetically clearly distinct lineages. In Pseudunela, sophisticated
microanatomy alone cannot reliably delimitate all of the
evolutionary lineages, but may reveal diagnosable differences among
pseudocryptic species once they have been delimited by molecular
analyses. Integrative taxonomy combining modern microanatomical
data on acochlidians with molecular analyses thus is superior to
individual approaches. With all key species revised in
microanatomical and testable detail, and many additional species
compared to such standard, now the Acochlidia probably range among
the best described heterobranch groups. There is, however, still a
critical gap of knowledge regarding biological observations and
ontogenetic stages. Future work also should focus on resolving the
exact origin of Acochlidia among Panpulmonata and on generating
comparative anatomical data from potential sister groups. In spite
of the urgency for speed facing the biodiversity crisis, my
dissertation showed the essential need for revisory work on
acochlidians, and this may be true also for other poorly known
micromolluscs. Integrative 3D microanatomical and molecular
approaches as exemplified herein are efficient, and thus suitable
to explore the diversity and evolution of neglected micromolluscs
within overall reasonable time scales.