There are some issues in the paper of Chiodin et al. that have to be commented:

1) In the results section the authors state that the female copulatory organ is anteriorly delimited by the bursal nozzle. This is wrong, the anterior-most part of the female copulatory organ is the vestibule or seminal receptacle which lies anterior to the bursal nozzle. This has been described in the original species description based on observations of live material, histological sections and electron microscopy [1]. Instructive pictures of live material can be seen in Figures 3 and 6 in [2] and micrographs of ultrathin sections from electron microscopy are available upon request.
2) The authors state that there is a pair of thick parenchymal muscles, which cross each other at a position dorsal to the mouth. First, no such muscles are apparent or marked in Figure 1C. Second, neither in the species description [1] nor an investigation of the musculature of adult specimens using fluorophore-tagged phalloidin and electron microscopy [3] such muscles have been shown or described. There are indeed muscles that form a cross pattern, however, they cross on the level of the statocyst [2,3,4,5]. Third, the authors state that hatchlings and juveniles have a very similar body plan (as adults) although they lack the reproductive organs and that the so-called cross muscles are likely used for feeding and egg laying. The cross-shaped ISH pattern, which, in accordance to the authors, marks “cross muscles” never occurs in hatchlings and juveniles – that would imply that they don’t eat.
How does this all fit? The authors themselves state in the discussion section in the right column of page 7 that the “cross muscle” genes are expressed in a very similar domain as IpPostHox [6]. A look at Figure 1B1 of the latter paper is very instructive and it must be stressed that the determination of the tissue that shows the X-shape as peripheral parenchyma has not only be inferred with WISH but also sections of WISH material (Figure C1 and C2 in [6]) and has been backed up by electron microscopy, a project in which I was personally involved. Of course there are muscles adjacent to the female copulatory organ [1] and it might be that we have missed such cells expressing IpPostHox. However, the authors do not discuss the conflict between the tissue that is inferred to express the genes in question (“cross muscles” vs. peripheral parenchyma) and their very similar domain with a single word.
3) In the original description Smith and Bush [1] denoted the positions of structures along the longitudinal body axis using a scale that represents percentage of body-length (U0-U100). The mouth is at U43 (slightly in front of the middle of the body), the bursa at U56 (slightly posterior of the middle of the body) and the male gonopore at U77. Applying these measurements it is striking that the labels for the mouth in the Figures 2H, I, O, 3O, and 5I are misplaced and actually mark the seminal bursa. Furthermore, the black arrow that should point to the female copulatory organ should be positioned further anterior in all the figures in which it has been applied. As is evident in Figure 5H the arrow points to the female sphincter, which indeed is part of the female tract but lies posterior to the seminal bursa, which, together with the vestibule and its tissue, is the central organ of the female copulatory organ and is evident in Figures 2T, 3B, H, and T (see discussion on the evolution of female copulatory organs in [2,7,8]).
4) The enzymatic ISH of IpTwist1 labels the cytoplasm of developing oocytes whereas the fluorescent ISH shows the nuclei of the oocytes strongly stained and some signal in the testes (Figure 4A,B; for instructive live images on the issue cytoplasm of oocytes vs. nuclei of oocytes see Figure 3 in [2]). It is remarkable that for IpTwist2 the case is exactly the other way around with regards to the enzymatic ISH and the fluorescent ISH. This would need some explanation.
5) In the discussion (page 7, left column) the authors write that all muscles express IpTrp. This is inconsistent with their notion of head-myocyte expression of IpmuscleLIM, IpPitx, IpFoxA2, IpMef2 and IpSix1/2. If IpTrp was expressed in all muscles an expression domain at the anterior end as in these genes (Figures 2B, H, 3B, N, T) should be expected in an IpTrp-ISH of adults and this is not the case (Figure 5H).


1. Smith JPS III, Bush L: Convoluta pulchra n. sp. (Turbellaria: Acoela) from
the east coast of North America. Transactions of the American Microscopical Society 1991, 110(1):12-26.
Available at: http://www.jstor.org/stab...
2. Achatz JG, Chiodin M, Salvenmoser W, Tyler S, Martinez P: The Acoela: on
their kind and kinships, especially with nemertodermatids and xenoturbellids (Bilateria incertae sedis). Org Div Evol 2012.
Doi: 10.1007/s13127-012-0112-4
3. Tyler S, Rieger RM: Functional morphology of musculature in the acoelomate worm, Convoluta pulchra (Plathelminthes). Zoomorphology 1999, 119(3):127-141.
Doi: 10.1007/s004350050087
4. Ladurner P, Rieger R: Embryonic muscle development of Convoluta pulchra (Turbellaria - Acoelomorpha, Platyhelminthes). Dev Biol 2000, 222(2):359-375.
Doi: 10.1006/dbio.2000.9715
5. Achatz JG, Martinez P: The nervous system of Isodiametra pulchra (Acoela) with a discussion on the neuroanatomy of the Xenacoelomorpha and its evolutionary implications. Front Zool 2012, 9:27.
Doi: 10.1186/1742-9994-9-27
6. Moreno E, De Mulder K, Salvenmoser W, Ladurner P, Martinez P: Inferring the ancestral function of the posterior Hox gene within the bilateria: controlling the maintenance of reproductive structures, the musculature and the nervous system in the acoel flatworm Isodiametra pulchra. Evolution & Development 2010, 12(3):258-266.
Doi: 10.1111/j.1525-142X.2010.00411.x
7. Achatz JG, Hooge M, Wallberg A, Jondelius U, Tyler S: Systematic revision of acoels with 9+0 sperm ultrastructure (Convolutida) and the influence of sexual conflict on morphology. J Zool Sys Evol Res 2010, 48(1):9-32. Doi: 10.1111/j.1439-0469.2009.00555.x
8. Jondelius U, Wallberg A, Hooge M, Raikova OI: How the Worm Got its Pharynx: Phylogeny, Classification and Bayesian Assessment of Character Evolution in Acoela. Systematic Biology 2011, 60(6),845-871.
Doi: 10.1093/Sysbio/Syr073