Insights into Ontogenetic Niche Changes in Bluegill, Lepomis macrochirus, Applying Combined Analyses of Stomach Content and Stable Isotopes

Round 1

Reviewer 1 Report

See comments in the attached draft.

Comments for author File: Comments.pdf

Author Response

We sincerely thank the reviewer #1 for his/her detail comments that would be help us to improve the quality of this manuscript. We tried to accept all comments, and below are the revision and answer for each comments

Indicate the study location, "... bluegill, Lepomis macrochirus, inhabiting the Yedang Reservoir in Korea".

• Line 15-16 is revised as “We integrated stomach content analysis (SCA) and stable isotope analysis (SIA) to understand ontogenetic niche shifts in the invasive freshwater fish, bluegill, Lepomis macrochirus, inhabiting the Yedang Reservoir in Korea”

Perhaps indicate the bias can occur with each of the component metrics (frequency, number, or bulk) used to calculate IRI. Ref #10 provides a good overview of these biases.

• We revised Line 51-52 as “Contents in a stomach which are partially digested and fragment of an organisms sometimes causes bias results between numerical and mass information [10]. Because IRI can reduce these errors in biased data, it has been widely applied in many studies on fish diets [11-13].”

Lines 58-62, this is a somewhat awkward sentence, consider revising

• It is revised to “The dietary information from SIA sometimes provides lower resolution than that from SCA, due to isotopic overlap between the primary producers occurring by isotopically homogeneous inorganic resources (i.e., dissolved inorganic carbon and nitrogen) in an ecosystem. However, time integrated dietary information as long as tissue turnover rate of a target organism obtained from SIA could supplement snap-shot information from SCA.”

 

Lines 76-78 awkward sentence with excessive use of commas,

• The sentence is corrected as “The bluegill Lepomis macrochirus, originates in North America, is a representative invasive species in Korea. The negative effects such as the extinction of native species in freshwater ecosystems caused by drastic increase of L. macrochirus population have long been noticed [33].”

Suggest modifying the sentence in lines 78-80 or adding a sentence that indicates the feeding placticity results from ontogenetic shifts in diet and habitat by bluegills as ontogeny is the premise of the current study (i.e., lines 88-94).

• We corrected as “ macrochirus is a highly predatory fish with a wide feeding niche, ranging from zooplankton to small vertebrates, with its feeding plasticity depending on its growth stage and the habitat it occupies.”

Diet preferences are determined by comparing the availability of prey to that used (i.e., consumed) by a predator. The present study is measuring prey use and therefore hypothesizing that prey consumption will be varied in accordance to growth.

• We understood what the reviewer is considering. Different diet consumption is not equal to preference. We agree with that point, and thus we changed “Diet preference to Diet consumption”

Seems like the location of the reservoir is the west-central region of the peninsula rather than the "middle".

• corrected

"...considered to be pelagic habitat."

• corrected

Spelling, "After"

• corrected

"...muscle tissue was excised, placed in a vial, and stored at -20 C."

• corrected

 

Some prey taxa were only identified to family or genera level so this sentence should read "...identified to the lowest taxonomic level by referring..."

• As reviewer’s comments, line 111-114 was revised to “Stomach contents were observed using a stereoscopic microscope and dietary items that were partially or wholly digested and morphologically difficult to recognize were identified to the lowest-taxonomic level by referring to illustrated books [40-42].”

IRI does not indicate prey preference, only the relative importance of prey consumed. The authors would need some measure of prey availability relative to that consumed by bluegills to establish any prey preferences.

• We revised to “The index of relative importance of each prey organisms was …”

Need to identify the symbols used in the sentence and equation, such as "Each isotope ratio was expressed with the conventional delta (δ) notation in parts per thousand (‰) using the following equation:"

• We revised to "Each isotope ratio was expressed with the conventional delta (δ) notation in parts per thousand (‰) using the following equation:"

"Differences in carbon and nitrogen isotopes for size classes of L. macrochirus were evaluated by..."

Revision.

Significant differences in carbon and nitrogen isotope ranges among size classified L. macrochirus were evaluated by one-way ANOVA

• We revised as reviewer’s comment “Differences in carbon and nitrogen isotopes for size classes of L. macrochirus were evaluated by one-way ANOVA, …”

This information is provided in the caption for Table 2 where letters indicate significant differences, so there is no need to include the statement in this paragraph.

• We accept the comment, and removed the sentence.

These are the totals for the study but what about sample sizes by size class for each sample site?

• We added Table 1 to show the number of samples by each size classes and sampling site.

 

Delete "The class of" as this is not a reference to the taxonomic level. Rather,modify the sentence to read "The most important prey groups for L. macrochirus were zooplankton..."

• Line 167-168 is revised as “The most important prey groups for L. macrochirus was zooplankton, …”.

Need to add more interpretation of the component diet metrics (number, mass, and frequency). Suggest modifying the sentence such as "The zooplankton Diaphanosoma brachyurum and Sindiaptomus sp. were numerically dominant, with correspondingly high importance values, while benthic macroinvertebrates in the family Chironomidae contributed the most to the total mass and had the highest importance value (Table1). These taxa were also the most frequently consumed by L. macrochirus. The smaller zooplankton are most numerous but contribute less to the total mass than relatively larger chironomids which, in turn, contribute less to the total number."

Additionally, the data show some of the classic biases with the component diet measures that were referenced in the Introduction, particularly bias arising from different sizes of prey items (see Liao et al., 2001, Transactions of the AMerican Fisheries Society https://doi.org/10.1577/1548-8659(2001)130%3C0583:EAOIOP%3E2.0.CO;2).

• Line 170 to 172 was revised as "The zooplankton Diaphanosoma brachyurum and Sindiaptomus sp. were numerically dominant, with correspondingly high importance values, while benthic macroinvertebrates in the family Chironomidae contributed the most to the total mass and had the highest importance value (Table1). These taxa were also the most frequently consumed by L. macrochirus. The smaller zooplankton are most numerous but contribute less to the total mass than relatively larger chironomids which, in turn, contribute less to the total number."

Not all of these identified to species level, change to "... these three taxa,..." Perhaps also indicate the other prey items contributed very little to the number, mass, and frequency, hence the negligible importance values

• Line 172 to 174 was revised as “Except for these three taxonomic gropus, the other prey items contributed very little to the number, mass, and frequency, hence indicated negligible importance values”

This is a family level identification, do not italicize (also Coenagrionidae and Corduliidae) and remove "sp."

• accepted

Again, IRI is not a measure of prey preference. Zooplankton were the most important prey item for the small fish group.

• “Preferred” is changed to “important”

shift to larger prey?

• The period was missing

Modify sentence as "...but medium to large fish did not exhibit the level of prey shifting observed at the other sites."

• Line 197 is corrected to “but medium to large fish did not exhibit the level of prey shifting observed at the other sites.”

The wording in this paragraph is confusing, is this comparison for mean values among size classes in all the study sites or, as indicated in Table 2, mean values within each study site? Please clarify as to what is actually being compared.

 If the comparison is among size classes for all the study sites, the test statistic and corresponding significance value need to be reported for each isotope.

If the comparison is within each site, the test statistics and significance values need to be reported for each isotope in each site.

Furthermore, the letter superscripts in Table 2 indicate significant differences for the carbon isotope, contrary to the statement in line 202.

• We tried compare isotope ranges among size classes at each. Line is a comparison of total ranges among study sites. However, this comparison is not necessary. We delete line 202.

There appears to be slight overlap for the ellipses of small and medium fish at site 2, unlike the clear separation for these groups at site 3.

• We added the results of overlapperd area estimated by SIBER and revised Line 222 to 226 as “The overlapped area of SEA between the small and medium group was 0.82 %² and 0.21 %² between small and large group at St. 1. On the other hand, the overlapped SEA of small group with other size classes in St. 2 and St. 3 was less than 0.001 %², indicating clearly separated isotopic niche. In comparison, the δ space between the medium and large groups within sampling sites partially overlapped (0.61 %² at St.1, 0.64 %² at St.2, and 0.25 %² at St.3, respectively), indicating niche competition between two of the TL groups.”

Delete this sentence as it is already established in the Materails and Methods and does not make for a good topic sentence for the paragraph.

• deleted

Suggest deleting "pelagic" to avoid confusion with th designation for site 3.

• deleted

"...whereas the IRI for zooplankton was higher for all sizes..."

• corrected

What were the "traits" for this site? Deeper water? Vegetative or other differences from littoral sites?

• 3 was more deeper water and less vegetation, and thus we demonstrated as pelagic habitat.

30. Suggestion

"..similar dietary composition between these two groups at each site,..."

• corrected

size class

• corrected

This statement needs to be modified as medium and large groups at site 3 did not have high IRI for benthic macroinvertebrates.

• Line 261-263 is revised as “In comparison, variation in the width and position of δ space shown in the medium and large size classes contrasts with their IRI in benthic macroinvertebrates.”

 

 

Dietary proportions were not similar as the medium group consumed some zooplankton but no fish and the large group consumed some fish but no zooplankton.

• We revised as “For example, large group of L. macrochirus at St. 1 and St. 2, adjacent to the inflow stream, showed different patterns of δ space in despite similar dietary proportions.”

There are other benthic dietary items listed in Table 1 but their importance was negligible. Modify sentence as "... which were the dominant benthic dietary items..."

• Revised to “This offset between the SCA and SIA results was derived from variable isotope ratios in benthic macroinvertebrates, particularly in chironomid larvae, which were the dominant benthic dietary items in our SCA results.”

This sentence is confusing. Were isotopic analyses conducted for dietary items in the current study but not included, or are the authors using data from other studies (refs. 49 and 50) to conclude isotopic variability for chironomids contributed to that of the fish in the current study. If the latter, delete the sentence in lines 268-270 and move the literature citations to the sentence in lines 265-268. Also modify the sentence in lines 265-268 as "... offset between SCA and SIA may have been derived...".

• Line 265-270 was revised as “This offset between the SCA and SIA results may have been derived from variable isotope ratios in benthic macroinvertebrates, particularly in chironomid larvae, were the dominant benthic dietary items in our SCA results .Unfortunately we did not include δ¹³C and δ¹⁵N analyses for diet items, but small spatial variation in chironomid larvae due to its short turnover rate and diverse larval stage has been reported [49, 50].”

This appears to be the case at sites 1 and 2 but not at site 3 where the ellipses of medium and large fish appear smaller than that of small fish, at least on the C13 axis.

• Expension is not a case for St. 3, but spatial variation is also included St .3

importance, not preference

• corrected

and otogeny.

• corrected

As indicated with prey preference, IRI is not indicative of selective feeding. One can only conclude that the relative importance of zooplankton was higher in smaller fish.

• As reviewer’s comment, our data is limited to discuss prey preference for the L. macrochirus. However, our opinion is that, based on our results and the related references, “limited diet consumption” in small size is more reliable than “importance”. Thus, we revised “Selective feeding behavior” to “narrow prey spectrum”

Again, medium and large fish at site 3 had relatively high IRI values for zooplankton which would suggest bluegills remain specialists in the pelagic environment. Also suggests habitat has a stronger influence on diet than otogeny.

• Our results showed regular diet consumption on small size class at both “pelagic” and “benthic” habitat, whereas medium and large size classes were showed habitat dependent diet consumption. In this point of view, we conclude the ontogenetic niche expansion (not diet shift) is the properties of this species.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

This is a nice short study. Still there are some major comments: a) ecological niche and isotopic niche are not the same, but the Authors use this term as such; b) sample numbers of groups for SIA are not shown in the manuscript, while this is important for quality control; and c) I suggest English to be checked once again. These are detailed below. And there are some additional minor comments, also listed below:

 

Line 19: IRI for invertebrates, not invertebrates themselves as it’s formulated now were high

 

Line 20 and throughout: ecological niche IS NOT equal to isotopic niche. I suggest Authors to read Hette-Tronquart, N. (2019) Isotopic niche is not equal to trophic niche. Ecology Letters 22, 1987–1989. DOI: 10.1111/ele.13218. As such, isotopic niche, not ecological niche is what the Authors are discussing here

 

Lines 24–25: repetition, importance of zooplankton for small fish is already mentioned above

 

Lines 27–28: this is well known fact, no need to state it again. Authors themselves state this is already known in Introduction, lines 70–73. It’s better to conclude about the study result

 

Lines 65–69: see above about differences of ecological and isotopic niches. These lines are a good place to introduce these differences in this manuscript

 

Lines 91–94: This sentence should be rewritten for clarity, and potentially splat to two sentences

 

Line 144: significant differences, not ‘significances’

 

Line 146: something like ‘p < 0.05’ taken as significant in this study’ would be better

 

Lines 96–150: sample number for SCA and for SIA, and how many small, medium and large fish there were for each method from each location – all this information is necessary in Materials and Methods, but is currently not mentioned in this chapter at all. I suggest adding this as a Table

 

Line 159: here it is recommended to cite this new Table from the comment above

 

Line 166–167: stomach contents were examined in all 235 fish, but the stomachs were empty in 19 fish

Please see above

Author Response

We sincerely thank the reviewer #2 for his/her useful comments that would be help us to improve the quality of this manuscript.

1. a) Ecological niche and isotopic niche are not the same, but the Authors use this term as such

• Reply: The authors agreed and appreciated the nice comments. As reviewer’s comments, we added the related sentences in line 70 “However, Isotopic niche estimation can lead to misunderstand for ecological interpretation due to properties of isotope signature in food chain such as variation in isotopic baseline and diverse trophic discrimination factor (Hette-Tronquart, 2019).” Thus, recently, …

1. b) Sample numbers of groups for SIA are not shown in the manuscript, while this is important for quality control

• We add a new table as below.

Table 1. Information on sample number for stomach contents analysis (SCA) and for stable isotope analysis (SIA), at each size classes and sampling sites

Size group (mm)	St.1	St.2	St.3	Total
Small (20-60)	28	42	24	94
Medium (61-99)	28	26	34	88
Large (100-163)	17	9	7	33
Total	73	77	65	215

 

1. c) I suggest English to be checked once again. These are detailed below. And there are some additional minor comments, also listed below:

• All minor and English editing comments are accepted and revised as below.

 

 

 

Minor comments

Line 19: IRI for invertebrates, not invertebrates themselves as it’s formulated now were high

• The sentence is revised as “Across all study sites, the index of relative importance (IRI) of zooplankton was high for small individuals, whereas those of benthic macroinvertebrates were high for the medium and large groups.”

Line 20 and throughout: ecological niche IS NOT equal to isotopic niche. I suggest Authors to read Hette-Tronquart, N. (2019) Isotopic niche is not equal to trophic niche. Ecology Letters 22, 1987–1989. DOI: 10.1111/ele.13218. As such, isotopic niche, not ecological niche is what the Authors are discussing here

• Ecological niche width is revised to “Isotopic niche width”.

Lines 24–25: repetition, importance of zooplankton for small fish is already mentioned above

• Instead of the sentence, we inserted that “Thus, together with the results on significantly high IRI of zooplankton. these individuals seem to have strong specialistic feeding ecology.”

Lines 27–28: this is well known fact, no need to state it again. Authors themselves state this is already known in Introduction, lines 70–73. It’s better to conclude about the study result

• We revised Lines 26-28 as “Our study demonstrates the applicability strength of combining SCA and SIA for ecological niche research with providing clear evidence of an ontogenetic niche shift in macrochirus and elucidates their feeding ecology.”

Lines 65–69: see above about differences of ecological and isotopic niches. These lines are a good place to introduce these differences in this manuscript

• We added following sentence in line 70 “However, isotopic niche estimation can lead to misunderstand for ecological interpretation due to properties of iosotope signals such as variation in isotopic baseline and diverse trophic discrimination factor (Hette-Tronquart, 2019).” Thus, recently …

Lines 91–94: This sentence should be rewritten for clarity, and potentially splat to two sentences

• We revised the line 90-94 as “We hypothesized that diet consumption and isotopic niche will be varied in accordance of both growth and habitat difference. In order to enhance understand such ontogenetic niche changes, our investigation was performed not only for different size classification, but also for different habitat condition such as pelagic and littoral environments.”

Line 144: significant differences, not ‘significances’

• We corrected it

Line 146: something like ‘p < 0.05’ taken as significant in this study’ would be better

• As another reviewer’s comment, we erase this sentence

Lines 96–150: sample number for SCA and for SIA, and how many small, medium and large fish there were for each method from each location – all this information is necessary in Materials and Methods, but is currently not mentioned in this chapter at all. I suggest adding this as a Table

• We add a new table as below.

Table 1. Information on sample number for stomach contents analysis (SCA) and for stable isotope analysis (SIA), at each size classes and sampling sites

Size group (mm)	St.1	St.2	St.3	Total
Small (20-60)	28	42	24	94
Medium (61-99)	28	26	34	88
Large (100-163)	17	9	7	33
Total	73	77	65	215

 

Line 159: here it is recommended to cite this new Table from the comment above

• We add a new table as comment.

Line 166–167: stomach contents were examined in all 235 fish, but the stomachs were empty in 19 fish

• Revised as suggested “Stomach contents were examined in all 235 individuals of macrochirus, but the stomachs were empty in 19 individuals (8.1%).

 

 

Author Response File: Author Response.pdf