Abstract
Nephridial (excretory organ) symbionts are widespread in lumbricid earthworms and the complexity of the nephridial symbiont communities varies greatly between earthworm species. The two most common symbionts are the well-described Verminephrobacter and less well-known Flexibacter-like bacteria. Verminephrobacter are present in almost all lumbricid earthworms, they are species-specific, vertically transmitted, and have presumably been associated with their hosts since the origin of lumbricids. Flexibacter-like symbionts have been reported from about half the investigated earthworms; they are also vertically transmitted. To investigate the evolution of this tri-partite symbiosis, phylogenies for 18 lumbricid earthworm species were constructed based on two mitochondrial genes, NADH dehydrogenase subunit 2 (ND2) and cytochrome c oxidase subunit I (COI), and compared to their symbiont phylogenies based on RNA polymerase subunit B (rpoB) and 16S rRNA genes. The two nephridial symbionts showed markedly different evolutionary histories with their hosts. For Verminephrobacter, clear signs of long-term host-symbiont co-evolution with rare host switching events confirmed its ancient association with lumbricid earthworms, likely dating back to their last common ancestor about 100 million years (MY) ago. In contrast, phylogenies for the Flexibacter-like symbionts suggested an ability to switch to new hosts, to which they adapted and subsequently became species-specific. Putative co-speciation events were only observed with closely related host species; on that basis, this secondary symbiosis was estimated to be minimum 45 MY old. Based on the monophyletic clustering of the Flexibacter-like symbionts, the low 16S rRNA gene sequence similarity to the nearest described species (<92%) and environmental sequences (<94.2%), and the specific habitat in the earthworm nephridia, we propose a new candidate genus for this group, Candidatus Nephrothrix.
Highlights
Earthworms of the family Lumbricidae have long been known to harbor extracellular symbiotic bacteria in their nephridia (Knop, 1926)
Fluorescence In Situ Hybridization (FISH) The localization of Verminephrobacter and Flexibacter-like bacteria in the nephridia was confirmed in two earthworm species; Eisenia andrei and Allolobophora chlorotica, using fluorescence in situ hybridization (FISH)
Fluorescence In Situ Hybridization (FISH) The presence of Verminephrobacter and Flexibacter-like bacteria was confirmed in Al. chlorotica and E. andrei (Figures 1B,C, respectively)
Summary
Earthworms of the family Lumbricidae have long been known to harbor extracellular symbiotic bacteria in their nephridia (Knop, 1926). The nephridia are the worm’s excretory system involved. The Ca. Nephrothrix-Verminephrobacter-earthworm symbiosis in excretion of nitrogenous waste and osmoregulation (Edwards and Bohlen, 1996). Nephrothrix-Verminephrobacter-earthworm symbiosis in excretion of nitrogenous waste and osmoregulation (Edwards and Bohlen, 1996) They are found in pairs in every segment, and each nephridium is coiled into three loops, where the symbionts reside in the ampulla part of the second loop (Figure 1A). Complexity and composition of the symbiont communities vary between worm species; few species host exclusively the betaproteobacterial Verminephrobacter, while most lumbricids have mixed nephridial communities (Davidson et al, 2010, 2013; Lund et al, 2010a). Verminephrobacter have been studied in detail during the past decade (for a recent review see Lund et al, 2014); these symbionts are species-specific (Schramm et al, 2003; Lund et al, 2010a), beneficial (Lund et al, 2010b), vertically transmitted via the cocoon (Davidson and Stahl, 2006, 2008), and presumably co-evolved with their host (Lund et al, 2010a)
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